Recombinant Chicken 39S ribosomal protein L51, mitochondrial (MRPL51)

What is MRPL51 and what is its primary function in mitochondria?

MRPL51 (mitochondrial ribosomal protein L51) is a 39S subunit protein of the mitochondrial ribosome. Mammalian mitochondrial ribosomal proteins are encoded by nuclear genes and facilitate protein synthesis within the mitochondrion. Mitochondrial ribosomes (mitoribosomes) consist of a small 28S subunit and a large 39S subunit, with MRPL51 being part of the latter . Unlike prokaryotic ribosomes, mammalian mitoribosomes have an estimated 75% protein to rRNA composition (the reverse ratio of prokaryotic ribosomes) and lack the 5S rRNA present in prokaryotic ribosomes .

MRPL51 primarily functions as a structural constituent of the mitochondrial ribosome, enabling proper assembly and function of the translation machinery that synthesizes proteins encoded by the mitochondrial genome. Its specific biochemical functions include protein binding and serving as a structural constituent of the ribosome .

How does recombinant chicken MRPL51 differ from human MRPL51?

While both chicken and human MRPL51 serve similar functional roles in mitochondrial translation, they exhibit species-specific sequence variations. These differences are significant when considering cross-species experimental designs or translational research. Sequence homology analysis between species shows that mitochondrial ribosomal proteins can differ substantially, which can affect their biochemical properties and interactions with other molecules .

When using recombinant chicken MRPL51 in research, investigators should be aware that:

• Protein sequence homology may be sufficient for studying conserved functions but might not perfectly model human-specific interactions

• Post-translational modifications may differ between species

• Protein-protein interaction networks might vary, potentially affecting experimental interpretation when studying complex formations

These differences must be considered when designing experiments using chicken MRPL51 as a model for human mitochondrial function or disease states.

What are the common expression systems used for recombinant MRPL51 production?

Several expression systems are commonly employed for the production of recombinant MRPL51, each with distinct advantages for different research applications:

Based on the search results, recombinant MRPL51 has been successfully expressed in multiple systems including mammalian cells, HEK293 cells, wheat germ, and in vitro cell-free systems . The choice of expression system depends on research objectives, with mammalian cells being preferred when post-translational modifications and proper folding are critical, while cell-free systems offer advantages for rapid production and when the protein might be toxic to host cells.

What protein tags are commonly used with recombinant MRPL51?

Several protein tags are employed with recombinant MRPL51 to facilitate purification, detection, and functional studies:

The data indicates that recombinant MRPL51 is commonly produced with various tags including His, GST, Avi, and Fc tags, or combinations thereof (such as His(Fc)-Avi-tagged variants) . The selection of an appropriate tag depends on the specific research application, with researchers needing to consider potential interference with protein function, requirements for downstream assays, and whether tag removal will be necessary.

How does MRPL51 contribute to mitochondrial function in cellular pathways?

• Mitochondrial translation initiation

• Mitochondrial translation elongation

• Mitochondrial translation termination

• Organelle biogenesis and maintenance

The protein interacts with several other mitochondrial ribosomal proteins and translation factors, including ICT1, MRPL50, and MRPL9 . These interactions are essential for proper mitochondrial translation and subsequent cellular energy production.

Gene Set Enrichment Analysis (GSEA) of tissues with differential MRPL51 expression has revealed associations with multiple pathways including 'DNA_REPAIR', 'UNFOLDED_PROTEIN_RESPONSE', 'MYC_TARGETS_V1', 'OXIDATIVE_PHOSPHORYLATION', 'MTORC1_SIGNALING', 'REACTIVE_OXYGEN_SPECIES_PATHWAY', 'MYC_TARGETS_V2', 'E2F_TARGETS', and 'G2M_CHECKPOINT' . These associations suggest that MRPL51 may have broader impacts on cellular function beyond its direct role in mitochondrial translation.

What role does MRPL51 play in cancer progression and metastasis?

Research has identified significant connections between MRPL51 expression and cancer progression, particularly in lung adenocarcinoma (LUAD). Studies demonstrate that:

• MRPL51 expression is upregulated at both mRNA and protein levels in LUAD tissues compared to normal lung tissues

• At the single-cell level, MRPL51 expression positively correlates with cell cycle progression, DNA damage response, DNA repair mechanisms, epithelial-mesenchymal transition (EMT), invasion, and proliferation of LUAD cells

• Knockdown of MRPL51 leads to:

  • Decreased N-cadherin and vimentin expression

  • Increased E-cadherin expression

  • G1 phase cell cycle arrest

  • Reduced cell invasion capacity

  • Slowed cell proliferation

The molecular mechanism involves FOXM1-mediated transcriptional activation of MRPL51 in LUAD, contributing to malignant tumor cell behaviors. This connection suggests that MRPL51 may represent a potential therapeutic target in cancer treatment strategies .

How can researchers effectively design knockdown experiments for MRPL51?

Designing effective MRPL51 knockdown experiments requires careful consideration of several methodological factors:

• Selection of knockdown technique:

  • shRNA-mediated knockdown delivered via lentiviral vectors has proven effective in multiple cell lines (A549 and Calu-3)

  • Multiple shRNA constructs should be tested (shMRPL51#1 and shMRPL51#2 have demonstrated efficient suppression)

• Validation of knockdown efficiency:

  • Western blotting for protein expression verification

  • RT-qPCR for mRNA expression quantification

  • Immunofluorescent staining for cellular localization assessment

• Functional assays following knockdown:

  • Flow cytometry to assess cell cycle distribution

  • CCK-8 assays to measure cell proliferation

  • Transwell invasion assays to evaluate invasive capacity

  • Western blotting for EMT markers (E-cadherin, N-cadherin, vimentin)

When implementing MRPL51 knockdown experiments, researchers should include appropriate controls and consider potential compensatory mechanisms, as mitochondrial function is critical for cell survival.

What are the regulatory mechanisms controlling MRPL51 expression?

The transcriptional regulation of MRPL51 involves specific transcription factors and promoter interactions. Research has identified FOXM1 as a key transcriptional activator of MRPL51:

• FOXM1-mediated regulation:

  • FOXM1 can bind directly to the MRPL51 gene promoter

  • This binding activates MRPL51 transcription

  • Dual luciferase assays using wild-type and mutant MRPL51 promoter sequences have confirmed this regulatory relationship

• Experimental approaches to study MRPL51 regulation:

  • Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) to validate transcription factor binding

  • Dual-luciferase reporter assays using wild-type and mutant MRPL51 promoter constructs

  • Analysis of MRPL51 expression following transcription factor knockdown

Understanding these regulatory mechanisms provides insights into how MRPL51 expression may be altered in disease states and offers potential targets for therapeutic intervention.

What are optimal protocols for recombinant MRPL51 purification?

Purification of recombinant MRPL51 requires optimization based on the expression system and tag selection. A general methodological approach includes:

• Expression system selection:

  • For structural studies: HEK293 cells with His-tag

  • For functional assays: Mammalian cells with appropriate tag

• Purification strategy:

  • Affinity chromatography using tag-specific resins (e.g., Ni-NTA for His-tagged proteins)

  • Size exclusion chromatography for higher purity

  • Ion exchange chromatography for removing contaminants with different charge properties

• Quality control assessments:

  • SDS-PAGE analysis for purity and expected molecular weight

  • Western blot for identity confirmation

  • Mass spectrometry for accurate mass determination and potential post-translational modifications

  • Circular dichroism for secondary structure verification

The purification protocol should be optimized based on downstream applications, with consideration for maintaining protein stability and function throughout the process.

How can researchers study MRPL51 interactions with other mitochondrial proteins?

Investigating MRPL51's interactions with other mitochondrial proteins requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-MRPL51 antibodies to pull down protein complexes

  • Identify interacting partners via mass spectrometry

  • Confirm specific interactions with western blotting

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins for in vivo proximity labeling

  • TurboID for rapid labeling of neighboring proteins

  • Mass spectrometry analysis of biotinylated proteins

• Yeast two-hybrid screening:

  • Use MRPL51 as bait to screen for interacting proteins

  • Validate interactions with alternative methods

• Pre-coupled magnetic beads:

  • Recombinant MRPL51 protein pre-coupled magnetic beads are available

  • Useful for pull-down assays to identify binding partners

• Structural biology approaches:

  • Cryo-EM for visualizing MRPL51 within the mitoribosome complex

  • X-ray crystallography for high-resolution structural data

Known interacting partners of MRPL51 include ICT1, MRPL50, and MRPL9 , which can serve as positive controls in interaction studies.

What assays are recommended for evaluating MRPL51's impact on mitochondrial translation?

To assess MRPL51's role in mitochondrial translation, researchers should consider these methodological approaches:

• Mitochondrial protein synthesis assays:

  • Pulse labeling with 35S-methionine in the presence of cytoplasmic translation inhibitors

  • Analysis of newly synthesized mitochondrially encoded proteins by SDS-PAGE and autoradiography

• Ribosome profiling:

  • Selective profiling of mitochondrial ribosomes

  • Analysis of ribosome positioning on mitochondrial mRNAs

  • Identification of translation efficiency changes

• Mitochondrial polysome profiling:

  • Fractionation of mitochondrial lysates on sucrose gradients

  • Analysis of ribosome distribution across fractions

  • Assessment of translation complex formation

• Functional readouts:

  • Oxygen consumption rate measurements

  • ATP production assays

  • Mitochondrial membrane potential assessment

  • Reactive oxygen species quantification

• MRPL51 mutation/deletion studies:

  • CRISPR-Cas9 generated mutants

  • Analysis of mitochondrial translation in MRPL51-deficient cells

  • Rescue experiments with wild-type MRPL51

These approaches should be tailored to the specific research question, with appropriate controls to ensure reliable interpretation of results.

How can researchers assess the clinical significance of MRPL51 expression in patient samples?

To evaluate the clinical relevance of MRPL51 expression in patient samples, researchers should implement the following methodological approaches:

What are common challenges in recombinant MRPL51 expression and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant MRPL51:

• Low protein yield:

  • Optimize codon usage for the expression system

  • Test different promoters and expression conditions (temperature, induction time)

  • Consider switching to a higher-yield expression system like HEK293 cells

  • Use fusion partners (GST, MBP) to enhance solubility

• Protein insolubility:

  • Modify buffer compositions (pH, salt concentration, detergents)

  • Express protein at lower temperatures (16-20°C)

  • Co-express with molecular chaperones

  • Consider cell-free expression systems for difficult-to-express constructs

• Protein instability:

  • Include protease inhibitors throughout purification

  • Optimize storage conditions (glycerol concentration, temperature)

  • Test stability-enhancing additives (trehalose, sucrose)

  • Consider Fc-fusion constructs for enhanced stability

• Incorrect folding:

  • Use mammalian expression systems for native-like folding

  • Implement stepwise refolding protocols if using inclusion bodies

  • Validate protein structure using circular dichroism or limited proteolysis

These challenges can often be resolved through systematic optimization of expression and purification conditions, guided by the specific downstream applications planned for the recombinant protein.

How should researchers interpret contradictory results in MRPL51 functional studies?

When faced with contradictory results in MRPL51 functional studies, researchers should:

• Evaluate experimental model variations:

  • Different cell lines may exhibit variable MRPL51 dependency

  • Species differences may influence functional outcomes

  • Primary cells vs. cell lines may show distinct responses

• Assess knockdown/knockout efficiency:

  • Partial vs. complete MRPL51 depletion may yield different phenotypes

  • Compensatory mechanisms may activate in complete knockout systems

  • Timing of analysis after MRPL51 depletion may influence results

• Consider context-dependent functions:

  • MRPL51 may have different roles depending on cellular stress conditions

  • Cell cycle stage may influence MRPL51 function

  • The role of MRPL51 in normal versus cancer cells may differ

• Scrutinize methodology differences:

  • Verify antibody specificity across studies

  • Compare protein tags used in different experimental setups

  • Evaluate the sensitivity of different assays measuring the same endpoint

• Perform integrative analysis:

  • Combine results from multiple experimental approaches

  • Consider conducting meta-analysis when applicable

  • Validate key findings using orthogonal methods

When studies report that MRPL51 knockdown affects EMT markers and cellular invasion , but other studies show different results, researchers should carefully examine the experimental conditions, cell types, and methodological differences that might explain these discrepancies.

What considerations are important when analyzing MRPL51 in different tissue types?

When analyzing MRPL51 across different tissue types, researchers should consider several important factors:

• Tissue-specific expression patterns:

  • Baseline MRPL51 expression varies across tissues

  • Mitochondrial content and function differs between tissue types

  • Energy demands influence mitochondrial protein expression

• Methodological adaptations:

  • Tissue-specific optimization of protein extraction protocols

  • Adjustment of antibody concentrations for immunohistochemistry

  • Consideration of tissue-specific housekeeping genes for normalization

• Physiological context:

  • Metabolic differences between tissues affect mitochondrial function

  • Tissue-specific interacting partners may modify MRPL51 function

  • Developmental stage may influence MRPL51 expression patterns

• Pathological significance:

  • MRPL51 dysregulation may have different consequences in different tissues

  • Cancer-specific changes in MRPL51 expression vary by tumor type

  • Correlation with prognosis may be tissue-specific

• Technical validation:

  • Confirm antibody specificity in each tissue type

  • Validate RNA expression with protein levels

  • Use multiple methodologies to confirm findings

Understanding these considerations is crucial when comparing MRPL51 function across different experimental models or when translating findings from one tissue type to another.

What are emerging areas of MRPL51 research with therapeutic potential?

Several promising research directions for MRPL51 have therapeutic implications:

• Cancer therapeutics:

  • Given the association between high MRPL51 expression and poor survival in lung adenocarcinoma , developing inhibitors targeting MRPL51 or its transcriptional regulators (such as FOXM1) could yield novel anti-cancer therapies

  • Combination approaches targeting MRPL51 and other mitochondrial translation components might provide synergistic effects

• Mitochondrial disease:

  • Exploring the role of MRPL51 in mitochondrial disorders could lead to new diagnostic markers

  • Gene therapy approaches to modulate MRPL51 expression might benefit patients with specific mitochondrial translation defects

• Metabolic regulation:

  • MRPL51's involvement in oxidative phosphorylation pathways suggests potential applications in metabolic disorders

  • Understanding how MRPL51 influences cellular energetics could inform treatments for conditions with impaired energy metabolism

• Cell stress responses:

  • The connection between MRPL51 and unfolded protein response pathways indicates potential roles in modulating cellular stress resistance

  • Therapeutic strategies targeting this relationship could enhance cellular resilience under pathological conditions

Future research should explore these directions while developing more specific tools to modulate MRPL51 function in a tissue-specific manner.

What technologies are advancing our understanding of MRPL51 structure and function?

Cutting-edge technologies are transforming MRPL51 research:

• Cryo-electron microscopy:

  • High-resolution structural studies of MRPL51 within the mitoribosome

  • Visualization of dynamic conformational changes during translation

  • Insights into MRPL51 interactions with other mitoribosomal components

• Single-cell technologies:

  • Single-cell RNA sequencing to reveal cell-specific roles of MRPL51

  • Single-cell proteomics to map protein interactions at individual cell level

  • Correlation of MRPL51 expression with cellular functional states

• CRISPR-based technologies:

  • Precise genome editing to study MRPL51 function

  • CRISPRi/CRISPRa for reversible modulation of MRPL51 expression

  • Base editing for introducing specific mutations to study structure-function relationships

• Computational approaches:

  • Machine learning to predict MRPL51 interactors

  • Molecular dynamics simulations to understand structural dynamics

  • Systems biology approaches to integrate MRPL51 into broader cellular networks

• Live-cell imaging:

  • Tracking MRPL51 dynamics in living cells

  • Visualizing mitochondrial translation in real-time

  • Correlating MRPL51 localization with mitochondrial function

These technologies promise to provide unprecedented insights into MRPL51 biology and potentially reveal new therapeutic targets.