Recombinant Oreochromis niloticus Guanine nucleotide-binding protein subunit beta-2-like 1 (gnb2l1)

What is GNB2L1/RACK1 in Nile tilapia and what cellular functions does it perform?

GNB2L1, commonly known as RACK1 (Receptor for Activated C Kinase 1), is a highly conserved scaffold protein in Nile tilapia (Oreochromis niloticus) with the gene ID 100534572 . This protein serves as an adaptor that interacts with multiple protein kinases and membrane receptors, enabling several crucial cellular functions including cyclin binding, enzyme binding, and protein domain-specific binding activities .

In Nile tilapia, as in other vertebrates, RACK1 is involved in multiple cellular processes including positive regulation of hydrolase activity, regulation of cellular protein metabolic processes, and modulation of signal transduction pathways . The protein is found in several cellular locations including the midbody, perinuclear region of cytoplasm, and phagocytic cup, suggesting its diverse roles in cellular compartments .

Interestingly, RACK1 has been identified as part of the IRE1-RACK1-PP2A complex, indicating its importance in cellular stress response pathways that may be particularly relevant for aquatic organisms that experience varying environmental conditions . Unlike some other scaffold proteins, RACK1 demonstrates constitutive expression during development and across multiple tissues in tilapia, suggesting its fundamental importance to basic cellular functions .

How does GNB2L1/RACK1 in Nile tilapia compare structurally to orthologs in other species?

The RACK1/GNB2L1 protein in Nile tilapia represents one of the most evolutionarily conserved scaffold proteins across species. Comparative analysis shows that the protein maintains high structural homology to its counterparts in other vertebrates including humans, rodents, and other fish species . The mRNA for Nile tilapia RACK1 is referenced as NM_001279516.1 with the protein reference NP_001266445.1, encoding a guanine nucleotide-binding protein subunit beta-2-like 1 .

The high degree of conservation is evident from cloning studies that have isolated complementary DNAs coding for RACK1 from both zebrafish (Danio rerio) and tilapia (Oreochromis niloticus), demonstrating constitutive developmental and tissue expression patterns that are similar across fish species . This conservation extends to the protein's functional domains, which typically include seven WD40 repeats that form a beta-propeller structure critical for protein-protein interactions.

The ORF nucleotide sequence of tilapia RACK1 spans 954 base pairs, which is comparable to the coding regions found in other vertebrates . This structural conservation underscores the fundamental importance of RACK1 in cellular processes that have been maintained throughout vertebrate evolution.

What are the recommended methods for detecting GNB2L1/RACK1 expression in Nile tilapia tissues?

Detection of GNB2L1/RACK1 expression in Nile tilapia tissues can be accomplished through several complementary techniques. Based on established protocols in tilapia research, the following methodological approaches are recommended:

RNA-based detection methods:

• RT-PCR using specific primers designed according to the GenBank sequence of Nile tilapia RACK1 (NM_001279516.1) . The primers should be designed to amplify a fragment of approximately 90-100 bp for optimal quantification.

• In situ hybridization using DIG-labeled probes to visualize tissue-specific expression patterns. This requires permeabilization with 0.2M HCl followed by proteinase K digestion, then hybridization with DIG-labeled probes (0.2 ng/1 ml of hybridization buffer) overnight at 50°C .

Protein-based detection methods:

• Immunohistochemistry using commercially available antibodies that cross-react with tilapia RACK1, followed by visualization with appropriate secondary antibodies such as biotinylated anti-mouse IgG (1:200) and detection with streptavidin-conjugated Alexa Fluor 488 .

• Western blotting using antibodies against conserved regions of RACK1, with β-actin as a loading control.

Recombinant protein expression:
For functional studies, the full-length ORF of tilapia RACK1 can be cloned into expression vectors such as pcDNA3.1-C-(k)DYK using seamless cloning technology . The recombinant protein can then be purified and used for various experimental applications including antibody production, protein interaction studies, and enzymatic assays.

How does GNB2L1/RACK1 participate in immune signaling in Nile tilapia compared to mammalian models?

GNB2L1/RACK1's role in immune signaling in Nile tilapia represents an intriguing area of comparative immunology. While mammalian RACK1 has been well-characterized as a scaffold protein involved in multiple immune signaling cascades, its specific functions in teleost fish like Nile tilapia show both conserved and divergent mechanisms.

In mammalian systems, RACK1 interacts with key components of immune signaling including protein kinase C (PKC) and has been implicated in the regulation of NF-κB pathways . In contrast, studies in Nile tilapia suggest that RACK1 may function alongside other immune regulatory proteins such as Sterile alpha and TIR motif-containing protein 1 (SARM1) . SARM1 has been identified as a negative regulator in anti-bacterial immune responses in Nile tilapia, functioning within the Toll-like receptor (TLR) signaling pathway .

The relationship between RACK1 and TLR signaling in tilapia appears to involve complex regulatory mechanisms that may differ from mammals. While mammalian SARM1 negatively regulates TRIF-dependent TLR signaling, the interactions between RACK1 and these immune regulatory components in tilapia require further investigation . This presents an opportunity for researchers to explore species-specific immune regulatory networks.

Methodologically, studying these interactions requires techniques such as co-immunoprecipitation, yeast two-hybrid assays, or modern proximity labeling approaches adapted for use in fish cell culture systems. Researchers should consider using recombinant Nile tilapia RACK1 protein as bait to identify novel interacting partners specific to teleost immune function.

What are the challenges and methodological considerations in studying GNB2L1/RACK1 promoter regulation in Nile tilapia?

Studying the promoter regulation of GNB2L1/RACK1 in Nile tilapia presents several technical challenges that require careful methodological consideration. Drawing from studies of the human GNB2L1 promoter, researchers should consider the following approaches and limitations:

Promoter characterization challenges:

• Identification of transcription start sites (TSS) in Nile tilapia RACK1 requires techniques such as 5' RACE (Rapid Amplification of cDNA Ends) or Fluorescently Labeled Oligonucleotide Extension (FLOE) . Based on human studies, researchers should anticipate the possibility of multiple alternative transcription start sites.

• Promoter mutation analysis requires the development of luciferase reporter constructs containing various lengths of the putative promoter region. This approach, similar to that used for human GNB2L1, would help identify minimal promoter elements sufficient for transcriptional activity .

Regulatory element considerations:

• In silico analysis should be performed to identify potential binding sites for transcription factors, including NF-κB (c-Rel) which has been implicated in the regulation of mammalian GNB2L1 .

• Treatment studies using compounds such as lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and steroid hormones should be conducted to assess their effects on promoter activity, as these have been shown to modulate GNB2L1 expression in human systems .

Experimental design for promoter studies:

• Cell culture systems derived from Nile tilapia are ideal, but challenging to establish. Researchers may need to adapt existing fish cell lines or develop primary cell cultures from tilapia tissues.

• Gateway cloning systems can be used to generate luciferase reporter constructs for promoter analysis, similar to the GW luc basic vector system used in human studies .

• Consideration should be given to distal regulatory elements (enhancers/silencers) that may lie outside the immediate promoter region, as human studies suggest that some regulators like DHEA may act through distant elements rather than the core promoter .

How can recombinant Nile tilapia GNB2L1/RACK1 protein be effectively produced and validated for structural and functional studies?

Production of functionally active recombinant Nile tilapia GNB2L1/RACK1 protein requires careful consideration of expression systems, purification strategies, and validation methods. The following methodological approach is recommended:

Expression system selection:

• Prokaryotic expression using E. coli BL21(DE3) may be suitable for structural studies but risks improper folding of the WD40 repeat domains characteristic of RACK1.

• Eukaryotic expression systems such as insect cells (Sf9 or Hi5) using baculovirus vectors are recommended for functional studies as they better maintain post-translational modifications and proper protein folding.

• For highest fidelity, consider fish cell line expression systems, though these typically yield lower protein quantities.

Cloning and expression strategy:

• The complete ORF sequence (954 bp) of Nile tilapia RACK1 should be cloned into an appropriate expression vector such as pcDNA3.1 with a C-terminal tag (e.g., DYKDDDDK/FLAG) to facilitate purification and detection .

• Seamless cloning technology is recommended to ensure accurate insertion without adding unwanted amino acids .

Purification protocol:

• Immobilized metal affinity chromatography (IMAC) using a histidine tag, followed by size exclusion chromatography to ensure homogeneity.

• For studies requiring higher purity, ion exchange chromatography may be employed as an additional step.

Validation methods:

• SDS-PAGE and Western blotting to confirm protein size and immunoreactivity.

• Circular dichroism (CD) spectroscopy to evaluate secondary structure, particularly important for WD40 repeat-containing proteins like RACK1.

• Functional validation through binding assays with known RACK1 interacting partners such as PKC.

• Thermal shift assays to assess protein stability under various buffer conditions.

The table below outlines key parameters for successful recombinant tilapia RACK1 production:

What is the relationship between GNB2L1/RACK1 and age-related immune function in teleost fish models?

Understanding the relationship between GNB2L1/RACK1 and age-related immune function in teleost fish requires examining parallels with mammalian systems while accounting for teleost-specific immune adaptations. In human leukocytes, research has demonstrated an age-dependent decline in RACK1 protein expression that correlates with plasma levels of dehydroepiandrosterone (DHEA) .

This age-related decline in RACK1 expression has been associated with defects in the protein kinase C (PKC) signal transduction pathway and related functional immune responses . The discovery that DHEA supplementation can counteract these age-associated defects in mammals suggests a potential regulatory mechanism that may also operate in teleost fish like Nile tilapia.

Methodological approaches for studying age-related RACK1 function in Nile tilapia:

• Developmental time-course studies measuring RACK1 expression across the lifespan of tilapia using qRT-PCR and Western blotting from various immune tissues including head kidney (equivalent to mammalian bone marrow).

• Comparison of immune challenge responses (e.g., bacterial pathogens) in young versus aged tilapia, with specific assessment of RACK1-dependent signaling pathways.

• Hormone manipulation studies using teleost-specific stress and sex hormones to determine if fish demonstrate similar hormonal regulation of RACK1 as observed with DHEA in mammals.

• Cell-specific expression analysis comparing RACK1 levels in sorted immune cell populations from fish of different ages.

Given that the promoter region of human GNB2L1 contains binding sites for glucocorticoid receptors and is modulated by cortisol , researchers should investigate whether similar regulatory mechanisms exist in Nile tilapia. This is particularly relevant as the balance between stress hormones and DHEA during aging might influence RACK1 expression and consequently immune function in fish as it does in mammals.

How does GNB2L1/RACK1 interact with other immune regulatory proteins like SARM1 in Nile tilapia innate immunity?

The potential interaction between GNB2L1/RACK1 and SARM1 (Sterile alpha and TIR motif-containing protein 1) represents an important area of research in Nile tilapia innate immunity. SARM1 has been identified as a negative regulator in anti-bacterial immune responses in Nile tilapia, playing a role in the Toll-like receptor (TLR) signaling pathway . Given that RACK1 functions as a scaffold protein for various signaling molecules, investigating its relationship with SARM1 could reveal important regulatory mechanisms in teleost innate immunity.

Methodological approaches for studying RACK1-SARM1 interactions:

• Co-immunoprecipitation assays: Using antibodies against either RACK1 or SARM1 to pull down protein complexes from Nile tilapia immune tissues or cell cultures, followed by Western blotting to detect the presence of the other protein .

• Proximity ligation assays (PLA): This technique can visualize protein-protein interactions in situ, allowing researchers to determine in which cell types and cellular compartments RACK1 and SARM1 might interact.

• Functional interaction studies: Examining how manipulation of RACK1 expression (via siRNA knockdown or overexpression) affects SARM1-dependent immune responses and vice versa.

• Pathogen challenge experiments: Assessing how bacterial infections alter the expression patterns and interactions between RACK1 and SARM1 in Nile tilapia immune tissues.

Since SARM1 is known to negatively regulate TRIF-dependent TLR signaling in mammals , researchers should specifically investigate whether RACK1 influences this regulatory function in Nile tilapia. This could be approached by studying downstream signaling events such as NF-κB activation and cytokine production in cells where RACK1 and/or SARM1 expression has been modified.

The table below outlines key experimental approaches for investigating RACK1-SARM1 interactions:

What expression systems are optimal for producing recombinant Nile tilapia GNB2L1/RACK1 for different research applications?

The choice of expression system for producing recombinant Nile tilapia GNB2L1/RACK1 depends critically on the intended research application. Different systems offer varying advantages in terms of protein folding, post-translational modifications, yield, and ease of purification. Based on the available data and established protocols, the following recommendations can be made:

Insect cell expression systems:
For functional studies requiring proper protein folding and some post-translational modifications, baculovirus-infected insect cells (Sf9, Hi5) represent an excellent compromise. The recombinant protein can be directed to include a secretion signal or be produced intracellularly with appropriate tags for purification.

Mammalian cell expression systems:
When studying interaction partners or conducting cellular assays, mammalian cell lines transfected with Nile tilapia RACK1 in vectors such as pcDNA3.1-C-(k)DYK provide a eukaryotic environment that may better preserve functional characteristics . While yields are typically lower, the protein is more likely to maintain native conformation.

Fish cell line expression:
For the highest biological relevance, expression in fish cell lines (though less commonly used) would provide the most appropriate cellular context. Available tilapia cell lines or other fish cell lines could be transfected with appropriate expression constructs.

The table below compares key parameters across different expression systems:

For most research applications, the insect cell system offers the best balance of yield and protein quality for recombinant Nile tilapia RACK1 production.

How should differential expression analysis of GNB2L1/RACK1 be conducted across tissues and developmental stages in Nile tilapia?

Conducting robust differential expression analysis of GNB2L1/RACK1 across tissues and developmental stages in Nile tilapia requires careful experimental design and appropriate analytical methods. Based on established protocols in fish gene expression studies, the following methodological framework is recommended:

Tissue sampling and developmental staging:

• For cross-tissue comparison, standardize sampling of at least 8-10 different tissues including brain (subdivided into forebrain, midbrain, and hindbrain), gill, heart, liver, kidney, spleen, intestine, muscle, and gonads .

• For developmental analysis, collect samples at key developmental milestones: early embryonic (cleavage, gastrulation), organogenesis, hatching, larval stages, juvenile, and adult (with age standardization).

RNA extraction and quality control:

• Extract total RNA using TRIzol reagent or commercial kits specifically validated for fish tissues.

• Assess RNA integrity using bioanalyzer technology (RIN > 8.0 recommended) and perform DNase treatment to remove genomic contamination.

Expression analysis methods:

• Quantitative RT-PCR: Design primers specific to Nile tilapia RACK1 (based on NM_001279516.1) with amplicon size of 90-100 bp. Use multiple reference genes (β-actin, EF1α, and 18S rRNA) for normalization according to MIQE guidelines .

• In situ hybridization: For spatial expression patterns, use DIG-labeled RNA probes designed against tilapia RACK1 following established hybridization protocols (50°C overnight hybridization, followed by stringent washing) .

• RNA-seq analysis: For genome-wide context, perform RNA-seq with a minimum depth of 20 million reads per sample. Analyze RACK1 expression in context of global transcriptomic changes.

Data analysis and visualization:

• For qPCR, calculate relative expression using the 2^-ΔΔCt method with appropriate statistical testing (ANOVA with post-hoc tests for multiple tissue comparison).

• For RNA-seq data, normalize read counts using TPM or FPKM methods, and employ DESeq2 or edgeR for differential expression analysis.

• Visualize expression patterns using heatmaps for cross-tissue comparison and line graphs for developmental trajectories.

• Perform co-expression network analysis to identify genes with similar expression patterns to RACK1 across tissues and developmental stages.

Validation approaches:

• Confirm RNA-level expression with protein-level analysis using Western blotting with antibodies verified to recognize Nile tilapia RACK1.

• Perform immunohistochemistry on tissue sections to corroborate in situ hybridization findings, using co-staining with cell-type specific markers such as HuC/D (neurons) or GFAP (glial cells) for detailed cellular localization .

How should researchers troubleshoot expression and purification issues with recombinant Nile tilapia GNB2L1/RACK1?

When working with recombinant Nile tilapia GNB2L1/RACK1, researchers may encounter various expression and purification challenges. The following systematic troubleshooting framework addresses common issues and their solutions:

Low expression levels:

• Problem: Poor codon optimization for expression host.
  Solution: Perform codon optimization of the 954 bp ORF sequence specifically for the expression system being used .

• Problem: Toxicity to host cells.
  Solution: Use tightly regulated inducible promoters (e.g., T7lac for E. coli) and reduce induction temperature to 16-20°C.

• Problem: mRNA instability.
  Solution: Check for rare codons or secondary structures in the mRNA that might affect translation efficiency.

Protein solubility issues:

• Problem: Formation of inclusion bodies in bacterial systems.
  Solution: Express as fusion proteins with solubility enhancers (MBP, SUMO, Thioredoxin) or switch to eukaryotic expression systems.

• Problem: Aggregation during purification.
  Solution: Include stabilizing agents (10% glycerol, 1mM DTT) in all buffers and avoid freeze-thaw cycles.

Purification challenges:

• Problem: Poor binding to affinity resins.
  Solution: Verify tag accessibility; consider moving the tag from C-terminus to N-terminus or using a longer linker sequence.

• Problem: Co-purification of contaminants.
  Solution: Implement a multi-step purification strategy combining affinity chromatography with ion exchange and size exclusion methods.

• Problem: Proteolytic degradation.
  Solution: Include protease inhibitor cocktails in all buffers and maintain samples at 4°C throughout purification.

Functional validation issues:

• Problem: Recombinant protein lacks expected activity.
  Solution: Verify proper folding using circular dichroism and thermal shift assays; ensure critical post-translational modifications are present.

• Problem: Inconsistent binding to known interaction partners.
  Solution: Test different buffer conditions and ensure that both proteins are in their native conformations.

The table below provides a decision tree for expression system selection based on troubleshooting outcomes:

What are the considerations for designing knockdown or knockout studies of GNB2L1/RACK1 in Nile tilapia?

Designing effective knockdown or knockout studies of GNB2L1/RACK1 in Nile tilapia requires careful consideration of the technical approaches, potential phenotypic outcomes, and appropriate controls. The following methodological framework outlines key considerations:

Technical approaches for gene manipulation:

• Morpholino knockdown: Suitable for embryonic studies, antisense morpholinos can be designed targeting the translation start site or splice junctions of tilapia RACK1 mRNA. Dose-dependent injection into fertilized eggs allows for titration of knockdown effects.

• CRISPR/Cas9 knockout: For permanent genetic modification, guide RNAs should be designed targeting early exons of the RACK1 gene based on the reference sequence (NM_001279516.1) . Multiple guide RNAs should be tested for efficiency.

• siRNA/shRNA approaches: For targeted studies in cell culture or specific tissues, RNA interference can be employed using sequences specific to tilapia RACK1.

Experimental design considerations:

• Complete vs. conditional knockout: Given RACK1's fundamental cellular roles, complete knockout may cause developmental lethality. Consider tissue-specific or inducible approaches using appropriate promoters.

• Developmental timing: If using inducible systems, carefully time the knockout to distinguish between developmental and physiological roles of RACK1.

• Dosage sensitivity: Design experiments to achieve varying levels of knockdown to identify potential threshold effects in RACK1-dependent functions.

Phenotype assessment strategies:

• Molecular phenotyping: Examine changes in key signaling pathways, particularly those involved in immune responses related to TLR signaling and interaction with SARM1 .

• Cellular phenotyping: Assess changes in cell proliferation, apoptosis, and morphology in tissues with high RACK1 expression.

• Immune challenge experiments: Test response to bacterial pathogens given RACK1's potential role in immune signaling alongside negative regulators like SARM1 .

Controls and validation:

• Off-target effect control: Include scrambled morpholinos/siRNAs or non-targeting CRISPR guides.

• Rescue experiments: Attempt phenotype rescue with wild-type RACK1 mRNA resistant to the knockdown approach.

• Validation of knockdown efficiency: Quantify RACK1 reduction at both mRNA (qRT-PCR) and protein (Western blot) levels.

• Heterozygote analysis: For CRISPR knockouts, analyze heterozygotes to assess dose-dependency of phenotypes.

The complex role of RACK1 as a scaffold protein interacting with multiple signaling pathways suggests that complete knockout might yield pleiotropic effects. Therefore, researchers should consider using tissue-specific or inducible approaches that allow for more precise dissection of RACK1 functions in specific contexts, particularly in immune responses where it may interact with regulatory proteins like SARM1 .

What are the future research directions for GNB2L1/RACK1 in Nile tilapia immunology and comparative biology?

The study of GNB2L1/RACK1 in Nile tilapia represents an important frontier in comparative immunology and teleost biology. Future research should focus on several promising directions to advance our understanding of this evolutionarily conserved scaffold protein in aquatic model organisms.

An immediate priority should be comprehensive characterization of RACK1's interactome in Nile tilapia immune cells using modern proteomics approaches. This would reveal species-specific interaction partners that might differ from the well-characterized mammalian RACK1 interactome. Particular attention should be paid to potential interactions with negative immune regulators like SARM1, which has been identified as important in Nile tilapia anti-bacterial immune responses .

The regulatory mechanisms controlling RACK1 expression in fish also warrant further investigation. While mammalian studies have identified age-dependent declines in RACK1 expression that correlate with hormone levels like DHEA , similar studies in fish models could reveal conserved or divergent regulatory mechanisms. The identification of two alternative transcription start sites in human GNB2L1 suggests complex transcriptional regulation that should be explored in tilapia.

From an evolutionary perspective, comparative studies examining RACK1 function across diverse fish species could provide insights into the evolution of immune signaling networks. The high conservation of RACK1 structure across vertebrates makes it an excellent candidate for studying how scaffold proteins maintain core functions while potentially adapting to species-specific signaling requirements.

Methodologically, the development of tilapia-specific cell lines and genetic tools would greatly facilitate these studies. The adaptation of CRISPR/Cas9 technology for tilapia, combined with tissue-specific or inducible promoter systems, would allow for precise manipulation of RACK1 expression in specific developmental contexts or immune cell populations.

Finally, translational research exploring how RACK1 signaling impacts disease resistance in aquaculture settings represents an important applied direction. Understanding how environmental stressors and pathogens modulate RACK1-dependent signaling could lead to improved management practices or selective breeding strategies to enhance disease resistance in this economically important fish species.

These research directions collectively would advance our fundamental understanding of comparative immunology while potentially yielding practical applications for sustainable aquaculture of Nile tilapia, one of the most important food fish species worldwide .

How can systematic integration of GNB2L1/RACK1 research in diverse species inform evolutionary understanding of scaffold protein functions?

Systematic integration of GNB2L1/RACK1 research across species, with particular focus on Nile tilapia within the broader evolutionary context, provides unique opportunities to understand the evolution of scaffold protein functions. RACK1 represents an ideal candidate for such comparative studies due to its remarkable conservation from worms to humans, with orthologs present in virtually all eukaryotes examined .

The integration of data from diverse species should follow a multi-layered approach examining:

Methodologically, this integration requires:

• Development of standardized protocols for interactome analysis across species

• Centralized databases compiling RACK1 interaction data from diverse organisms

• Phylogenetic methods to trace the evolution of specific RACK1 functions

• Functional assays adaptable across species to test conserved activities