Recombinant Pongo abelii 39S ribosomal protein L42, mitochondrial (MRPL42)

What is the function of mitochondrial ribosomal proteins in primates?

Mitochondrial ribosomal proteins are essential components of mitoribosomes involved in protein synthesis within the mitochondria. In primates like Pongo abelii (Sumatran orangutan), these proteins are crucial for translating mitochondrially encoded genes involved in oxidative phosphorylation and energy production. Research on human mitochondrial ribosomal proteins has shown that they contribute to mitochondrial function through protein synthesis in the mitochondrion . Their conservation across species suggests similar functional importance in Pongo abelii, with potential species-specific adaptations. Disruption of mitochondrial ribosomal proteins can lead to mitochondrial dysfunction and has been implicated in various pathological conditions.

How is recombinant MRPL42 typically produced for research applications?

Recombinant MRPL42 for research purposes is commonly produced using bacterial expression systems, particularly E. coli. Based on protocols for similar mitochondrial ribosomal proteins, the process typically involves:

• Cloning the MRPL42 gene from Pongo abelii cDNA into a suitable expression vector

• Transforming the construct into E. coli expression strains

• Inducing protein expression under optimized conditions

• Protein purification using affinity chromatography (commonly His-tag based systems)

• Quality control validation through SDS-PAGE and Western blotting

Similar to the production method for related ribosomal proteins, the tag type is often determined during the manufacturing process to optimize yield and purity . The recombinant protein typically achieves >85% purity as determined by SDS-PAGE, similar to other mitochondrial ribosomal proteins .

What are the recommended storage conditions for recombinant MRPL42?

Based on guidelines for similar mitochondrial ribosomal proteins, recombinant MRPL42 should be stored according to the following recommendations:

• Lyophilized form: 12 months stability at -20°C/-80°C

• Liquid form: 6 months stability at -20°C/-80°C

• Working aliquots: Store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they may compromise protein integrity

For reconstitution, it is recommended to centrifuge the vial briefly before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol as a cryoprotectant for long-term storage . The default final concentration of glycerol is typically 50% for optimal stability.

How does MRPL42 expression impact cellular function and what knockdown studies reveal?

Studies on human MRPL42 provide insights that may be relevant to the Pongo abelii ortholog. When MRPL42 is knocked down in human glioma cells (U251 and A172), the following effects have been observed:

• Significant suppression of cell proliferation in multiple cell lines

• Cell cycle arrest predominantly in G₁ and G₂/M phases

• Decrease in S-phase cell population

• Activation of apoptosis pathways

• Increased caspase-3/caspase-7 activity

These findings suggest that MRPL42 functions as a potential oncogene in certain human cellular contexts, where its silencing blunts proliferation and activates apoptosis mechanisms . When designing studies on Pongo abelii MRPL42, researchers should consider these potential functional impacts while accounting for species-specific differences.

What experimental approaches are most effective for studying MRPL42 function in comparative primate studies?

For comparative studies between human and Pongo abelii MRPL42, consider the following experimental approaches:

• Sequence homology and phylogenetic analysis:

  • Conduct sequence alignment between human and Pongo abelii MRPL42

  • Analyze conserved domains and species-specific variations

  • Construct phylogenetic trees to understand evolutionary relationships

• Expression profiling:

  • qRT-PCR to quantify expression levels across tissues

  • RNA-seq for transcriptome-wide analysis

  • Comparison to reference data such as TCGA databases for human tissues

• Functional studies:

  • Lentiviral shRNA knockdown in cellular models

  • CRISPR-Cas9 genetic modification

  • Rescue experiments with species-specific variants

  • High-content screening assays for proliferation analysis

  • Flow cytometry for cell cycle distribution analysis

  • Caspase activity assays for apoptosis assessment

What are the challenges in interpreting ortholog data between human and Pongo abelii MRPL42?

When interpreting data across species, researchers should be aware of the following challenges:

• Evolutionary divergence: Despite high sequence similarity, functional differences may exist between human and Pongo abelii MRPL42 due to evolutionary adaptations.

• Interaction network variations: MRPL42 interacts with species-specific protein networks that may differ between humans and orangutans, potentially affecting functional outcomes.

• Expression pattern differences: Tissue-specific expression patterns may vary between species, impacting the interpretation of functional studies.

• Ortholog group complexity: As demonstrated in ortholog databases like InParanoiDB, mitochondrial proteins often belong to multiple ortholog groups with varying confidence scores, complicating direct comparisons .

• Experimental model limitations: Human cell lines may not accurately represent Pongo abelii cellular environments, necessitating careful validation of interspecies extrapolations.

What experimental controls should be included when studying recombinant MRPL42?

When designing experiments with recombinant Pongo abelii MRPL42, incorporate the following controls:

• Negative controls:

  • Non-targeting shRNA constructs for knockdown studies

  • Empty vector controls for overexpression studies

  • Vehicle-only treatments for drug interaction studies

• Positive controls:

  • Well-characterized mitochondrial proteins with known functions

  • Human MRPL42 for comparative analyses

  • Established inducers of mitochondrial stress or dysfunction

• Validation controls:

  • Multiple shRNA constructs targeting different regions to confirm specificity

  • Rescue experiments with shRNA-resistant constructs

  • qRT-PCR and Western blot validation of knockdown efficiency

• Technical controls:

  • Include multiple cell lines to control for cell type-specific effects

  • Time-course experiments to capture temporal dynamics

  • Dose-dependent analyses for quantitative assessment

How can researchers optimize cell-based assays to study MRPL42 function?

Based on successful approaches with human MRPL42, consider these optimization strategies:

• Cell proliferation assessment:

  • High-content screening (HCS) assays provide quantitative data on cell growth over time

  • MTT assays offer complementary measures of metabolic activity

  • Time-course experiments (1-5 days) capture the full growth curve dynamics

• Cell cycle analysis:

  • Flow cytometry with propidium iodide staining for DNA content

  • EdU incorporation assays for S-phase quantification

  • Synchronization protocols to examine specific cell cycle phases

• Apoptosis detection:

  • Annexin V/PI staining for early and late apoptotic cell quantification

  • Caspase-3/7 activity assays using fluorogenic substrates

  • TUNEL assays for DNA fragmentation detection

• Mitochondrial function:

  • Oxygen consumption rate measurements

  • Mitochondrial membrane potential assays

  • ATP production quantification

  • ROS detection methods

What approaches can be used to identify MRPL42 interaction partners?

To characterize the interactome of Pongo abelii MRPL42:

• Co-immunoprecipitation (Co-IP):

  • Use tagged recombinant MRPL42 to pull down interacting proteins

  • Validate interactions with reciprocal Co-IP experiments

  • Employ mass spectrometry for unbiased identification of binding partners

• Proximity labeling:

  • BioID or APEX2 fusion proteins for labeling proteins in close proximity

  • Particularly useful for capturing transient or weak interactions in mitochondria

• Yeast two-hybrid screening:

  • Modified for mitochondrial proteins to identify direct protein-protein interactions

  • Validate hits with orthogonal methods

• Crosslinking mass spectrometry:

  • Chemical crosslinking followed by MS analysis to map interaction interfaces

  • Provides structural insights into protein complexes

How can comparative studies of MRPL42 contribute to understanding mitochondrial evolution?

Comparative analysis of MRPL42 across species can provide valuable insights into mitochondrial evolution:

• Evolutionary rate analysis:

  • Calculate evolutionary rates of MRPL42 compared to other mitoribosomal proteins

  • Identify signatures of positive or purifying selection

  • Map conservation patterns to functional domains

• Structure-function relationships:

  • Compare structural properties of MRPL42 across species

  • Correlate structural variations with functional adaptations

  • Use homology modeling to predict species-specific structural differences

• Mitoribosome assembly:

  • Investigate species-specific differences in mitoribosome assembly

  • Examine co-evolution patterns with interacting proteins

  • Assess evolutionary constraints on protein-protein and protein-RNA interfaces

Ortholog databases show that Pongo abelii mitochondrial proteins participate in multiple ortholog groups with varying bitscores and inparalog scores, suggesting complex evolutionary relationships that merit further investigation .

What potential role does MRPL42 play in disease pathogenesis based on current evidence?

Based on studies of human MRPL42:

• Cancer biology:

  • MRPL42 is significantly upregulated in glioma tissues compared to normal tissues

  • Knockdown experiments demonstrate its importance for cancer cell survival

  • Cell cycle arrest and apoptosis induction suggest potential therapeutic targets

• Mitochondrial disorders:

  • As a component of mitoribosomes, MRPL42 dysfunction may contribute to mitochondrial translation defects

  • Could potentially impact energy production in high-energy demanding tissues

  • May be involved in mitochondrial stress responses

• Evolutionary medicine:

  • Comparing disease associations across species could reveal conserved pathogenic mechanisms

  • Species-specific adaptations might explain differential disease susceptibility

  • Pongo abelii-specific variants could provide insights into primate-specific disease mechanisms

What novel technical approaches might advance MRPL42 research?

Emerging technologies that could enhance MRPL42 research include:

• Cryo-EM studies:

  • High-resolution structural analysis of species-specific mitoribosomes

  • Visualization of MRPL42 within the mitoribosomal complex

  • Identification of conformational changes during protein synthesis

• Single-cell technologies:

  • Single-cell RNA-seq to capture cell-type specific expression patterns

  • Spatial transcriptomics to map tissue distribution with high resolution

  • Single-cell proteomics for protein-level analysis

• Organoid models:

  • Development of primate-specific organoid systems

  • Comparison of MRPL42 function in human vs. Pongo abelii organoids

  • Disease modeling in physiologically relevant 3D systems

• CRISPR genome editing:

  • Humanization of MRPL42 in model organisms

  • Introduction of Pongo abelii-specific variants in human cells

  • Precise manipulation of regulatory elements to study expression control

What is known about the sequence characteristics of Pongo abelii MRPL42?

While the search results don't provide the specific sequence for Pongo abelii MRPL42, we can consider the information available for related mitochondrial ribosomal proteins:

For accurate sequence information, researchers should consult the UniProt database for the most current Pongo abelii MRPL42 sequence data.

What cell-based assay results might be expected when studying MRPL42 function?

Based on human MRPL42 studies, researchers might expect similar patterns when investigating Pongo abelii MRPL42:

These expected outcomes are extrapolated from human studies and should be validated specifically for Pongo abelii MRPL42 in appropriate experimental systems.

How does ortholog analysis inform our understanding of MRPL42 conservation?

Ortholog analysis from databases like InParanoiDB provides insights into evolutionary relationships: