Recombinant Mouse CDK5 regulatory subunit-associated protein 1 (Cdk5rap1)

What is the primary molecular function of Cdk5rap1?

Cdk5rap1 (CDK5 regulatory subunit-associated protein 1) functions as a radical SAM (S-adenosylmethionine) enzyme that catalyzes the conversion of N6-isopentenyladenosine (i6A) into 2-methylthio-N6-isopentenyladenosine (ms2i6A) in RNA molecules . While initially identified as an inhibitor of CDK5 kinase (preventing formation of the activated CDK5 complex), more recent research has established its primary role as a methylthiotransferase that deposits 2-methylthio modifications on mitochondrial tRNAs . This modification enables efficient intramitochondrial translation and is essential for proper mitochondrial function, especially under stress conditions.

How does Cdk5rap1 differ from CDKAL1, and what are their respective roles?

Despite belonging to the same family of methylthiotransferases, Cdk5rap1 and CDKAL1 (CDK5 regulatory subunit-associated protein 1-like 1) have distinct functions:

• Subcellular localization: Cdk5rap1 functions primarily in mitochondria, while CDKAL1 is an endoplasmic reticulum-resident protein .

• Physiological roles: Cdk5rap1 deficiency leads to mitochondrial dysfunction and accelerated cellular senescence, particularly affecting hearing function . CDKAL1 is associated with type 2 diabetes susceptibility, functioning in pancreatic β-cells to enhance translational fidelity of proinsulin transcripts .

• Target specificity: While both modify tRNAs, they exhibit different substrate preferences, with CDKAL1 specifically enhancing translational fidelity of proinsulin transcripts .

What phenotypes are observed in Cdk5rap1-knockout mouse models?

Cdk5rap1-knockout mice exhibit several distinct phenotypes that provide insights into the protein's physiological roles:

• Accelerated hearing loss: Female Cdk5rap1-KO mice display early-onset hearing loss phenotypically similar to age-related hearing loss (AHL) from 12 weeks of age, with significantly elevated auditory brainstem response (ABR) thresholds across all tested frequencies .

• Cochlear degeneration: Progressive loss of outer hair cells (OHCs), inner hair cells (IHCs), and spiral ganglion cells (SGCs) occurs earlier than in control mice .

• Cellular senescence: Increased senescence-associated β-galactosidase (SA-β-gal) activity is observed in multiple cochlear structures, including the organ of Corti, spiral ligament, spiral ganglion cells, and stria vascularis from as early as 4 weeks of age .

• Metabolic alterations: Significant changes in TCA cycle metabolites, including decreased fumarate and increased pyruvate and lactate levels, indicating mitochondrial dysfunction .

The following table summarizes ABR threshold differences between Cdk5rap1-KO and control mice:

How should researchers design studies to characterize cellular senescence in Cdk5rap1-deficient models?

When investigating cellular senescence in Cdk5rap1-deficient models, implement the following methodological approaches:

• Comprehensive tissue mapping: Perform SA-β-galactosidase staining across multiple cochlear structures at various time points. In Cdk5rap1-KO mice, senescent cells appear earlier in the organ of Corti, spiral ligament, spiral ganglion cells, and stria vascularis compared to controls .

• Quantitative analysis: Calculate the cochlear SA-β-gal-positive ratio using image analysis. Research shows progressive increases in Cdk5rap1-KO mice:

  • 4 weeks: 0.008 in KO vs. 0.002 in control (P<0.001)

  • 12 weeks: 0.016 in KO vs. 0.002 in control (P<0.001)

  • 20 weeks: 0.03 in KO vs. 0.008 in control (P<0.001)

• Complementary senescence markers: Confirm findings using additional markers such as lipofuscin accumulation (detectable with H&E staining) which typically appears in the same areas as SA-β-gal activity .

• Senescence-associated secretory phenotype (SASP) analysis: Measure inflammatory cytokines and other SASP components to characterize the senescent cell secretome.

What are the optimal methods for studying Cdk5rap1 methylthiotransferase activity in vitro?

To study Cdk5rap1 methylthiotransferase activity effectively:

• Enzyme preparation:

  • Express recombinant Cdk5rap1 in E. coli systems with an N-terminal His-tag for purification

  • Ensure >90% purity via SDS-PAGE validation

  • Verify protein integrity using circular dichroism or thermal shift assays

• Reaction components:

  • Substrate: Synthesized or purified tRNAs containing N6-isopentenyladenosine (i6A)

  • Cofactors: S-adenosylmethionine (SAM) as the methyl donor

  • Iron-sulfur cluster components: Fe2+ and sulfide sources

  • Reducing agents: Dithiothreitol (DTT) or β-mercaptoethanol

  • Buffer: Optimal pH (typically 7.0-8.0) with physiological salt concentration

• Detection methods:

  • HPLC separation of modified nucleosides after enzymatic digestion of tRNA

  • Mass spectrometry to detect and quantify ms2i6A formation

  • Thin-layer chromatography with radiolabeled substrates

• Control reactions:

  • Include catalytically inactive Cdk5rap1 mutants

  • Use non-i6A-containing tRNAs as negative controls

  • Run reactions with and without SAM to confirm SAM-dependent activity

What techniques are recommended for assessing mitochondrial dysfunction in Cdk5rap1-deficient models?

To thoroughly evaluate mitochondrial dysfunction in Cdk5rap1-deficient models:

• Metabolomic profiling:

  • Measure TCA cycle intermediates using LC-MS/MS

  • Focus on fumarate (decreased in Cdk5rap1-KO), pyruvate, and lactate (both increased in Cdk5rap1-KO)

  • Conduct principal component analysis to identify metabolic shifts

• Mitochondrial respiratory function:

  • Oxygen consumption rate (OCR) measurements using Seahorse XF analyzers

  • Complex-specific respiratory analysis with appropriate substrates and inhibitors

  • ATP production rate assays

• Mitochondrial translation efficiency:

  • 35S-methionine pulse-labeling to measure mitochondrial protein synthesis rates

  • Northern blot analysis of ms2i6A-modified tRNAs

  • Ribosome profiling to detect translational pausing or frameshifting

• Oxidative stress assessment:

  • ROS detection using fluorescent probes

  • Mitochondrial membrane potential measurements

  • Antioxidant enzyme activity assays

How can researchers investigate the relationship between Cdk5rap1-mediated tRNA modifications and age-related hearing loss?

To investigate this relationship:

• Time-course experiments:

  • Measure tRNA modification levels using mass spectrometry at different ages

  • Correlate modification changes with onset of hearing deficits

  • Track progression of cellular senescence markers

• Cochlear function assessments:

  • Combine ABR measurements with distortion-product otoacoustic emissions (DPOAE) testing

  • Measure endocochlear potential (EP) at different ages (significantly lower in KO mice at 12 weeks: P=0.001; 20 weeks: P<0.001)

  • Assess Na+/K+-ATPase α1 expression in the spiral ligament, which shows significant reduction in KO mice from 4 weeks of age

• Cell-type specific analyses:

  • Use cochlear explant cultures from Cdk5rap1-KO mice

  • Perform single-cell RNA sequencing to identify vulnerable cell populations

  • Employ cell-specific Cre drivers for conditional knockout studies

• Intervention studies:

  • Test antioxidant treatments to counteract mitochondrial dysfunction

  • Investigate senolytic compounds to eliminate senescent cells

  • Develop gene therapy approaches to restore Cdk5rap1 function

What approaches are recommended for resolving contradictory findings about Cdk5rap1's dual roles?

When addressing the apparent contradiction between Cdk5rap1's initially described role as a CDK5 inhibitor and its established function as a methylthiotransferase:

• Structure-function analysis:

  • Generate domain-specific mutants to determine if different protein regions mediate distinct functions

  • Use co-immunoprecipitation assays to identify interaction partners in different cellular compartments

  • Perform subcellular fractionation to track protein localization

• Temporal considerations:

  • Investigate whether Cdk5rap1's functions vary during different cellular states

  • Examine potential developmental shifts in protein function

  • Assess whether stress conditions alter its primary role

• Cell-type specificity:

  • Compare Cdk5rap1 function in neuronal versus non-neuronal cells

  • Investigate tissue-specific expression patterns and interaction partners

  • Use cell-type specific conditional knockouts to assess differential phenotypes

• Mechanistic connections:

  • Investigate whether mitochondrial dysfunction affects CDK5 activity

  • Examine if CDK5-mediated phosphorylation regulates Cdk5rap1 methylthiotransferase activity

  • Study feedback mechanisms between these apparently distinct pathways

How might findings from Cdk5rap1-knockout mice inform therapeutic approaches for age-related hearing loss?

Translating findings from Cdk5rap1-knockout models to therapeutic development:

• Targeting mitochondrial dysfunction:

  • Develop mitochondria-targeted antioxidants to reduce oxidative stress

  • Investigate compounds that enhance mitochondrial biogenesis

  • Design therapies that improve mitochondrial tRNA modification in the absence of functional Cdk5rap1

• Senescence-based interventions:

  • Test senolytic drugs that specifically eliminate senescent cells in cochlear structures

  • Evaluate senomorphic compounds that suppress the senescence-associated secretory phenotype

  • Determine optimal therapeutic windows based on cochlear SA-β-gal-positive ratio progression

• Genetic approaches:

  • Develop AAV-based gene therapy vectors for cochlear-targeted Cdk5rap1 delivery

  • Investigate CRISPR-based approaches to correct pathogenic CDK5RAP1 mutations

  • Explore RNA therapeutic strategies to enhance residual Cdk5rap1 activity

• Biomarker development:

  • Identify blood-based biomarkers that reflect cochlear mitochondrial dysfunction

  • Develop imaging approaches to detect cochlear senescence in vivo

  • Establish predictive models for age-related hearing loss based on genetic and metabolic profiles

What methodological approaches should be used to investigate potential connections between Cdk5rap1 and neurodegenerative diseases?

To explore potential connections between Cdk5rap1 and neurodegenerative diseases:

• Comparative analysis:

  • Investigate Cdk5rap1 expression and activity in neurodegenerative disease models

  • Compare tRNA modification patterns in affected brain regions

  • Assess whether CDK5 dysregulation (implicated in Alzheimer's disease) correlates with Cdk5rap1 dysfunction

• Mechanistic studies:

  • Evaluate whether Cdk5rap1 deficiency affects tau phosphorylation (a key process in Alzheimer's disease)

  • Investigate mitochondrial dysfunction as a common pathway in neurodegeneration and Cdk5rap1-deficiency

  • Examine whether restoring Cdk5rap1 function ameliorates neurodegenerative phenotypes

• Human genetic studies:

  • Perform targeted sequencing of CDK5RAP1 in patients with early-onset neurodegeneration

  • Investigate potential genetic interactions between CDK5RAP1 and known neurodegeneration-associated genes

  • Conduct genome-wide association studies to identify CDK5RAP1 variants associated with disease risk

• Translational models:

  • Develop human iPSC-derived neuronal models with CDK5RAP1 mutations

  • Use brain organoids to study the impact of Cdk5rap1 deficiency on neural development

  • Test candidate therapeutic approaches in multiple model systems