PTRH2 Human

What is PTRH2 and what are its primary cellular functions?

PTRH2 (Peptidyl-tRNA Hydrolase 2), also known as BIT1 (Bcl-2 inhibitor of transcription 1), is an evolutionarily highly conserved protein that belongs to the peptidyl-tRNA hydrolase family . Its primary function is to release peptidyl moieties from tRNA, preventing the accumulation of prematurely dissociated peptidyl-tRNA which could inhibit protein synthesis and be toxic to cells . Beyond this canonical role, PTRH2 has multiple functions including:

• Regulation of cell survival and death mechanisms

• Promotion of cell survival through integrin-mediated signaling

• Regulation of muscle differentiation during human development

• Modulation of anoikis (cell death triggered by detachment from extracellular matrix)

The protein is expressed in all tissue types and plays a crucial role in normal development .

Where is PTRH2 localized within human cells?

PTRH2 demonstrates multi-compartmental localization within human cells. Current research indicates that the 19 kDa protein (179 residues, UniProt ID: Q9Y3E5) is present in multiple cellular locations:

• Mitochondrial outer membrane (primary location)

• Plasma membrane

• Endoplasmic reticulum

• Golgi apparatus

This diverse localization pattern explains its involvement in various cellular functions. Researchers can visualize PTRH2 localization through immunofluorescence techniques using specific antibodies against PTRH2, combined with organelle markers .

What signaling pathways involve PTRH2?

PTRH2 participates in several critical signaling pathways that regulate cell survival, growth, and differentiation:

Methodologically, researchers typically employ Western blotting to measure the phosphorylation status of these pathway components in both wild-type and PTRH2-deficient cells .

What mutations in the PTRH2 gene are associated with IMNEPD and how do they correlate with clinical phenotypes?

Research has identified several mutations in the PTRH2 gene associated with IMNEPD. The known mutations include:

The clinical manifestations vary in frequency among patients:

• Most common (>80%): Motor delay (92%), neuropathy (90%), distal weakness (86.4%), intellectual disability (84%), hearing impairment (80%), ataxia (79%)

• Less common (30-70%): Head/face deformities (~70%), hand deformity (64%), cerebellar atrophy/hypoplasia (47%), pancreatic abnormality (35%)

• Least common (<30%): Diabetes mellitus (~30%), liver abnormality (~22%), hypothyroidism (16%)

Disease severity appears to correlate with the type of mutation, with research suggesting that missense mutations may result in milder phenotypes compared to frameshift or nonsense mutations that completely abolish protein function .

How can researchers design experiments to evaluate the functional impact of PTRH2 mutations?

When investigating the functional consequences of PTRH2 mutations, researchers should consider a multi-tiered experimental approach:

• Cellular models:

  • Establish patient-derived fibroblasts or Epstein-Barr virus-transformed lymphocytes (LCLs)

  • Create isogenic cell lines with specific PTRH2 mutations using CRISPR-Cas9 gene editing

  • Analyze cellular PTRH2 localization through immunofluorescence

  • Measure cell viability, proliferation, and apoptosis rates

• Biochemical analyses:

  • Quantify protein levels of PTRH2 and downstream effectors (pAKT/AKT, pMAPK/MAPK, pFAK/FAK, Bcl-2, pS6) via Western blotting

  • Assess mitochondrial respiratory chain enzyme activities in patient fibroblasts using established protocols

  • Evaluate protein-protein interactions through co-immunoprecipitation

• Functional assays:

  • Analyze integrin-mediated cell adhesion and anoikis

  • Study effects on muscle differentiation

  • Assess impact on protein synthesis through metabolic labeling

• Gene expression analysis:

  • Perform quantitative real-time PCR to measure expression levels of PTRH2 and related genes

  • Consider RNA-seq to identify global transcriptional changes

These methodological approaches can help elucidate how specific mutations affect PTRH2 function and contribute to disease pathogenesis.

What animal models are available for studying PTRH2 deficiency?

The primary animal model for studying PTRH2 deficiency is the Ptrh2 knockout mouse. Key features of this model include:

• Generation method: Ptrh2-mutant mice were generated by breeding Ptrh2flox/flox mice with MORE-Cre mice (B6.129S4-Meox2tm1(cre)Sor/J) to obtain heterozygote Ptrh2flox/− MeoxCre/− mice, which were subsequently backcrossed and intercrossed to obtain Ptrh2-mutant mice with a pure C57BL/6 genetic background .

• Genotyping: Performed using specific primers (G2F 5′-TGG GTC TTT GAA TCA ACT AG-3′, G1R 5′-ACA TGC CAC AAG CAA CTC CA-3′, and 30d 5′-TTT GAG ACC CTA TCA CTC CAC ACG-3′), yielding a 250 bp band in wild-type, a 200 bp band in homozygous Ptrh2 mutant mice, and both bands in heterozygous mice .

• Phenotype: Homozygous Ptrh2 mutants develop a "runting" (dystrophy) syndrome postnatally and die within the first 2 weeks of life , recapitulating some features of human IMNEPD.

• Analysis methods:

  • Monitoring development and behavior

  • Histological analysis of organs (brain, liver, pancreas, muscle, diaphragm)

  • Measurement of elastase activity in stool

  • Quantitative analysis of mRNA and protein expression

This model has been instrumental in understanding the physiological role of PTRH2 and the pathological consequences of its deficiency.

How does PTRH2 interact with the mTOR pathway and what are the implications for cell size regulation?

PTRH2 has been linked to the mechanistic target of rapamycin (mTOR) pathway, which is central to cell growth and size regulation. Current research indicates:

• Pathway interaction: PTRH2 appears to modulate mTOR signaling, potentially through its interactions with other signaling molecules like AKT .

• Methodological approach: Researchers can assess this interaction by:

  • Measuring phosphorylation of mTOR substrates such as S6 ribosomal protein (pS6)

  • Analyzing effects of PTRH2 deficiency on cell size in various tissues

  • Examining impacts on protein synthesis rates

  • Investigating potential rescue of phenotypes with mTOR pathway modulators

• Findings from patient cells: Analysis of fibroblasts from IMNEPD patients has demonstrated altered mTOR signaling, supporting PTRH2's role in this pathway .

• Experimental considerations: When investigating this interaction, researchers should control for nutrient conditions, as mTOR signaling is highly sensitive to amino acid and growth factor availability.

Understanding this relationship may provide insights into the growth retardation phenotype observed in IMNEPD patients and open potential therapeutic avenues.

What methodological approaches can be used to study PTRH2's role in muscle differentiation?

PTRH2 has been implicated in myogenic differentiation, making this an important area for researchers investigating IMNEPD's neuromuscular manifestations. Key methodological approaches include:

• In vitro myogenesis models:

  • Utilize C2C12 myoblast cell lines with PTRH2 knockout/knockdown

  • Establish primary myoblast cultures from patient biopsies or Ptrh2-mutant mice

  • Induce differentiation and monitor fusion index, myotube formation, and myogenic marker expression

• Molecular analysis:

  • Assess expression of myogenic regulatory factors (MyoD, Myogenin, MRF4)

  • Analyze PTRH2 expression during different stages of muscle differentiation

  • Investigate PTRH2's interaction with muscle-specific transcription factors

• Signaling pathway analysis:

  • Examine the impact on PI3K-AKT signaling, which is crucial for muscle development

  • Investigate effects on calcium signaling and mechanotransduction

  • Study potential cross-talk with other pathways important for myogenesis

• Histological examination:

  • Perform immunohistochemistry on muscle sections from Ptrh2-mutant mice

  • Analyze fiber type composition, size, and ultrastructure

  • Assess neuromuscular junction formation and integrity

These methodologies can help elucidate why patients with PTRH2 mutations experience distal muscle weakness and other neuromuscular symptoms.

How can researchers analyze the impact of PTRH2 mutations on anoikis and cell attachment mechanisms?

PTRH2 is a key player in anoikis, a form of programmed cell death induced when cells detach from the extracellular matrix. To study this relationship:

• Cell detachment assays:

  • Culture cells on poly-HEMA-coated plates to prevent attachment

  • Measure survival rates of control versus PTRH2-mutant cells in suspension

  • Analyze apoptotic markers (Annexin V, caspase activation)

• Cellular localization studies:

  • Track PTRH2 translocation from mitochondria to cytosol upon detachment

  • Use immunofluorescence to visualize PTRH2 phosphorylation and complex formation with AES (amino-terminal enhancer of split)

  • Employ subcellular fractionation techniques followed by Western blotting

• Integrin signaling analysis:

  • Measure focal adhesion complex formation

  • Assess FAK phosphorylation status and downstream signaling

  • Evaluate integrin expression and activation patterns

• In vivo implications:

  • Investigate tissue integrity in Ptrh2-mutant mice, focusing on tissues with high cell turnover

  • Examine embryonic development stages where cell migration and attachment are critical

These approaches can provide insights into how PTRH2 mutations affect cellular attachment and survival mechanisms, potentially explaining certain developmental and neurological features of IMNEPD.

What techniques are recommended for detecting and characterizing novel PTRH2 variants in undiagnosed patients?

For researchers screening undiagnosed patients with symptoms suggestive of IMNEPD, a systematic approach to PTRH2 variant detection is recommended:

• Primary genetic screening:

  • Whole-exome sequencing (WES) followed by bioinformatic analysis focusing on homozygous or compound heterozygous variants

  • Sanger sequencing of the PTRH2 gene (NM_016077) for confirmation

  • Consider using panels for neurogenetic disorders that include PTRH2

• Variant interpretation:

  • Utilize prediction tools such as SIFT, PolyPhen2, and MutationTaster to assess potential pathogenicity

  • Check variant frequency in population databases (Exome Variant Server, 1000 Genome database, dbSNP138)

  • Search for the variant in disease-specific databases

• Segregation analysis:

  • Test family members to establish inheritance patterns

  • Create pedigrees to visualize segregation of the variant with disease

• Functional validation:

  • Establish patient-derived cell lines to assess PTRH2 expression and function

  • Consider in silico structural modeling to predict the impact on protein structure

  • Design minigene assays if splicing effects are suspected

This multi-tiered approach can help identify and characterize novel PTRH2 variants in patients with compatible clinical presentations.

Table: Clinical Manifestations in PTRH2-Associated IMNEPD

Data compiled from literature review

Table: Known PTRH2 Mutations and Their Characteristics

What are the most promising future research directions for PTRH2-related disorders?

Based on current knowledge, several research directions show particular promise:

• Therapeutic development:

  • Investigation of mTOR pathway modulators to address growth and neurological symptoms

  • Exploration of gene therapy approaches for PTRH2 replacement

  • Development of compounds that might enhance residual PTRH2 activity in missense mutations

• Genotype-phenotype correlations:

  • Expansion of patient cohorts to better understand the clinical spectrum

  • Detailed analysis of how different mutations affect protein function and clinical outcome

  • Creation of mutation-specific cellular and animal models

• Basic biology:

  • Further elucidation of PTRH2's role in diverse cellular processes

  • Investigation of tissue-specific requirements for PTRH2

  • Study of potential modifiers that might explain phenotypic variability

• Diagnostic improvements:

  • Development of biomarkers for early detection

  • Creation of functional assays to assess PTRH2 activity in patient samples

  • Incorporation of PTRH2 in neurodevelopmental disorder screening panels