Recombinant Mouse Retrotransposon-derived protein PEG10 (Peg10), partial

What is the evolutionary origin of mouse PEG10?

PEG10 is a mammalian gene derived from the domestication of retrotransposable elements. It belongs to the class of genes that originated from long terminal repeat (LTR) retrotransposons/retroviruses that were integrated into the mammalian genome during evolution . The protein exhibits homology with LTR retrotransposon GAG and POL proteins, containing structural elements that reflect its viral ancestry . PEG10 is therian-specific, meaning it is found only in placental mammals and marsupials, suggesting its acquisition occurred after the divergence from monotremes but before the marsupial-placental mammal split .

What are the primary protein domains in mouse PEG10?

Mouse PEG10 contains two major protein isoforms produced through a frameshift mechanism typical of retroviruses:

• RF1 PEG10 (Gag-like protein): Encoded by reading frame 1, this corresponds to the first 325 amino acid residues and contains domains similar to retroviral Gag proteins .

• RF1/RF2 PEG10 (Gag-Pol-like polyprotein): Encoded by both reading frames 1 and 2, this longer protein (626 amino acids) contains both the Gag-like domain and additional Pol-like domains including an aspartic protease domain .
  The protease domain contains an -Asp-Ser-Gly- sequence, which corresponds to the consensus -Asp-Ser/Thr-Gly- active-site motif characteristic of retroviral aspartic proteases .

How is PEG10 gene expression regulated in mice?

PEG10 is a paternally expressed imprinted gene, meaning only the paternal allele is expressed while the maternal allele is silenced through epigenetic mechanisms . This imprinting is critical for normal development. In research contexts, it's important to note that when studying PEG10 function through genetic manipulation, only paternally transmitted mutations will manifest phenotypes due to this imprinting pattern . Expression studies should always consider this unique regulatory feature when designing experiments or interpreting results.

How does the protease domain of PEG10 affect its biological function?

The aspartic protease domain of PEG10 appears to play a crucial role in its biological function. Studies using site-directed mutagenesis of the active site (D370A mutation) have demonstrated that this domain mediates autoproteolytic processing of the PEG10 polyprotein . When the protease active site is mutated, the full-length RF1/RF2 PEG10 protein accumulates without being processed into separate domains, indicating impaired self-cleavage activity .
Functionally, the protease activity significantly impacts cellular processes. Research shows that cells expressing PEG10 with intact protease activity exhibited increased proliferation but decreased viability compared to cells expressing protease-inactive mutants . This suggests that PEG10 protease regulates the balance between cell proliferation and cell death, potentially contributing to its roles in development and disease.

What is the frameshift mechanism in PEG10 translation?

PEG10 utilizes a −1 ribosomal frameshift mechanism that is characteristic of retroviruses. This process occurs during translation and allows the production of two protein products from a single mRNA :

• RF1 PEG10: Produced when ribosomes translate only reading frame 1

• RF1/RF2 PEG10: Produced when ribosomes shift from reading frame 1 to reading frame 2 at a specific "slippery" sequence
  The frameshift occurs at a G-GGA-AAC heptanucleotide sequence that serves as a "slippery" site . This mechanism typically has low efficiency, resulting in more RF1 protein than RF1/RF2 protein, which is confirmed by Western blot analysis showing predominance of the RF1 form in cell lysates .
  Researchers can experimentally manipulate this frameshift by inserting a single adenine into the slippery sequence (G-GGA-AAA-C), which forces translation into the -1 frame and increases production of the RF1/RF2 polyprotein .

How does PEG10 form virus-like particles and what cellular functions do they serve?

PEG10 can assemble into endogenous virus-like particles (eVLPs) due to its retroviral Gag-like domain. Studies have shown that overexpression of PEG10 in HEK293FT cells results in the formation of extracellular vesicles containing the capsid (CA) domain in the culture medium . These particles can transport molecules between cells, similar to how retroviruses package and transfer genetic material.
In brain tissue, PEG10 has been identified as a component of stress granules and extracellular vesicles responsible for carrying mRNAs and proteins for intercellular communication . This vesicle-forming ability has been leveraged in experimental contexts to engineer PEG10-based VLPs for delivering cargo molecules, including antigens for vaccine development .
Unlike viral VLPs, PEG10-derived endogenous VLPs lack strong immunogenicity, which can be advantageous or disadvantageous depending on the application. For vaccine development, this limitation has been addressed by modifying the particles with immunostimulatory molecules like CpG oligonucleotides to enhance their ability to activate dendritic cells .

What expression systems are recommended for producing recombinant mouse PEG10?

For recombinant mouse PEG10 production, several expression systems have been successfully employed:

• HEK293T Cell Expression: This mammalian expression system is widely used for PEG10 studies as it provides proper eukaryotic post-translational modifications and processing . The human embryonic kidney 293T cell line has low endogenous PEG10 expression, making it suitable for overexpression studies.

• Commercial Recombinant Proteins: For researchers seeking purified protein, commercial sources offer recombinant mouse PEG10 with various tags (such as His-Fc-Avi) expressed in HEK293 cells . These products typically guarantee ≥85% purity by SDS-PAGE and endotoxin levels below 1.0 EU per μg .

• Vector Selection: The pQE-TriSystem expression vector has been successfully used for PEG10 cloning and expression . When cloning PEG10, it's important to note that the cDNA sequence contains an XhoI restriction site that may need to be mutated to facilitate cloning .
  For optimal expression and purification, consider incorporating affinity tags such as polyhistidine tags and including protease cleavage sites (such as thrombin) to allow tag removal after purification .

What are the key methodological considerations when designing PEG10 mutants?

When designing PEG10 mutants to study its function, consider the following methodological approaches:

• Active Site Mutations: For studying protease function, the D370A mutation in the aspartic protease domain's active site (-Asp-Ser-Gly- motif) effectively inactivates the enzyme while preserving protein structure . This mutation results in accumulation of unprocessed polyprotein.

• Frameshift Modifications: To study the functions of RF1/RF2 polyprotein, insertion of a single adenine into the G-GGA-AAC "slippery" heptanucleotide sequence (to G-GGA-AAA-C) forces translation in the -1 frame, increasing production of the RF1/RF2 protein without relying on natural frameshift efficiency .

• Domain Fusion Strategies: For creating PEG10-antigen fusions (e.g., for vaccine development), neoantigens or epitopes can be fused to the C-terminal of the PEG10 gag domain . This approach has been used successfully to create VLPs carrying antigenic cargo.

• Validation Methods: Always validate mutants through sequencing and functional assays. For protease mutants, Western blotting can confirm the accumulation of unprocessed polyprotein . For VLP-forming mutants, electron microscopy and extracellular vesicle isolation protocols can confirm particle formation .

What techniques are available for detecting and quantifying PEG10 expression?

Several methods have been validated for detecting and quantifying PEG10:

• ELISA Assays: Commercial mouse PEG10 ELISA kits are available with high sensitivity (0.072 ng/mL) and detection ranges of 0.156-10 ng/mL . These kits are validated for measuring PEG10 in mouse serum, plasma, and cell culture supernatants with intra-assay and inter-assay CV values of 5.3% and 7.6%, respectively .

• Western Blotting: PEG10 can be detected using Western blot with specific antibodies. It's important to note that some antibodies may only target RF1 PEG10 (1-325 residues) and will not detect RF2 PEG10 alone . When performing Western blot for PEG10, expected molecular weights are approximately 40 kDa for RF1 PEG10 and 75 kDa for processed products of RF1/RF2 PEG10 .

• PCR-Based Methods: For mRNA expression analysis, RT-PCR and qPCR can be employed. Due to PEG10's imprinted status, RNA-based methods should be complemented with protein detection to confirm functional expression .

• Immunohistochemistry: For tissue localization studies, immunohistochemical staining using anti-PEG10 antibodies has been successfully employed to determine expression patterns in placental tissues and other organs .

How does PEG10 protease activity affect placental development in mice?

The protease activity of PEG10 plays a critical role in placental development. Studies of mice with mutations in the viral aspartic protease motif in the POL-like region (PEG10-ASG mice) revealed distinct phenotypes compared to complete PEG10 knockout . While PEG10 knockout mice exhibit early embryonic lethality due to severe placental defects, protease mutant mice show:

• Perinatal lethality (approximately 50% dead fetuses at 18.5 dpc) rather than early embryonic lethality

• Fetal and placental growth retardation starting at mid-gestation

• Severe defects in fetal vasculature within the placenta
  Biochemical analysis confirmed that the protease mutation prevented the normal self-cleavage of PEG10, as evidenced by the absence of the ~75 kDa cleavage product in mutant placentas . This demonstrates that PEG10's protease activity is essential for normal placental vascular development and fetal growth, but not for the earliest stages of placental formation.
  The expression pattern of PEG10 in the three trophoblast cell layers surrounding fetal capillary endothelial cells suggests its protease activity may regulate factors involved in vascular development or remodeling .

What experimental evidence supports PEG10's role in cellular proliferation?

Multiple lines of experimental evidence demonstrate PEG10's influence on cellular proliferation:

• Cell Proliferation Assays: Transfection of cells with frameshift mutant fsRF1/RF2 PEG10 (which forces expression of the full-length polyprotein) resulted in significantly increased cellular proliferation compared to controls, particularly in HEK293T cells .

• Protease Dependence: When the protease active site was mutated (D370A), the proliferation-enhancing effect was diminished, indicating that the protease activity is essential for PEG10's growth-promoting function .

• Cell Viability Effects: Interestingly, while enhancing proliferation, expression of wild-type PEG10 with active protease significantly decreased cell viability by >60% . This effect was reversed when the protease was inactivated, suggesting PEG10 may regulate a balance between proliferation and cell death.

• Cell Type Specificity: The effects of PEG10 on proliferation appear to be cell-type dependent, with different responses observed in various cell lines, indicating context-specific regulation or function .
  These findings collectively suggest that PEG10, particularly through its protease activity, can promote cell proliferation but may simultaneously regulate cell viability, potentially reflecting its evolutionary origin from retroviruses that must balance host cell proliferation with viral replication.

How can PEG10 be targeted for studying specific disease mechanisms?

Given PEG10's implications in both developmental processes and disease states, several approaches can be used to target it for mechanistic studies:

• Genetic Modification Models: The comparison between complete PEG10 knockout mice (early embryonic lethal) and protease domain mutants (perinatal lethal) provides a powerful system for distinguishing between structural and enzymatic functions .

• Cell-Specific Expression Analysis: In disease contexts, particularly cancers where PEG10 is often overexpressed, cell type-specific expression analysis can identify where and when PEG10 activity contributes to pathogenesis .

• Signaling Pathway Interactions: PEG10 has been shown to inhibit TGF-beta signaling by interacting with the TGF-beta receptor ALK1 . This interaction can be targeted through co-immunoprecipitation studies or signaling pathway reporter assays to understand disease-specific effects.

• Protease Inhibitor Development: The retroviral-like aspartic protease domain presents a potential drug target. Structure-based design of specific inhibitors against PEG10 protease might offer therapeutic approaches for conditions where PEG10 activity is pathologically elevated .

• VLP-Based Delivery: The ability of PEG10 to form virus-like particles can be leveraged not only for studying its function but also for developing therapeutic delivery systems targeting specific tissues or cell types .

How can PEG10-assembled VLPs be engineered for vaccine development?

PEG10's ability to form endogenous virus-like particles (eVLPs) presents a unique platform for vaccine development, particularly for cancer immunotherapy. The engineering process involves several key considerations:

• Antigen Fusion Design: Neoantigens or target epitopes can be genetically fused to the C-terminal of the PEG10 gag domain . This has been demonstrated with multiple antigens, including Hepa1-6 cell-derived neoantigens and HBc18-27 epitopes .

• Immunogenicity Enhancement: Since PEG10-derived eVLPs lack intrinsic immunogenicity compared to viral VLPs, they can be modified with immunostimulants like CpG oligonucleotides (CpG-ODN) . CpG-ODN acts as a TLR9 agonist and enhances dendritic cell activation.

• Targeting Strategies: To improve uptake by antigen-presenting cells, CpG-ODN modification leverages binding to DEC-205 receptors on dendritic cells, enhancing both uptake through pinocytosis and subsequent immune activation .

• Production Methods: Transfection of HEK293T cells with PEG10-antigen fusion constructs has been successfully used for eVLP production . The resulting particles can be isolated from culture medium and characterized for size, antigen content, and immunogenicity.
  This approach offers several advantages over traditional vaccines, including the ability to co-deliver both antigen and adjuvant in a single particle, enhanced targeting to antigen-presenting cells, and the flexibility to incorporate various neoantigens for personalized cancer immunotherapy .

What methodological approaches can reveal PEG10's role in RNA transport?

PEG10 has been implicated in RNA transport through its association with extracellular vesicles and stress granules in the brain . To investigate this function, researchers can employ several methodologies:

• RNA-Immunoprecipitation (RIP): This technique can identify RNA species associated with PEG10 in various cellular contexts. By immunoprecipitating PEG10 and analyzing bound RNAs through sequencing, specific cargo RNAs can be identified.

• Extracellular Vesicle Isolation: Differential ultracentrifugation, size exclusion chromatography, or commercial EV isolation kits can be used to isolate PEG10-containing vesicles from culture medium or biological fluids . The RNA content of these vesicles can then be analyzed.

• Fluorescent RNA Tracking: By labeling specific RNAs and co-expressing them with wild-type or mutant PEG10, their packaging into VLPs and intercellular transfer can be visualized and quantified using microscopy techniques.

• Stress Granule Analysis: Since PEG10 associates with stress granules , techniques such as immunofluorescence co-localization studies during cellular stress conditions can reveal dynamic interactions with RNA-binding proteins.

• Domain Mutation Studies: Creating targeted mutations in RNA-binding regions of PEG10 can help elucidate which domains are responsible for RNA selection and packaging into VLPs.
  These approaches can provide insights into how PEG10-mediated RNA transport contributes to intercellular communication in normal development and disease states.

How does proteomic analysis inform the study of PEG10 interactions and regulation?

Proteomic approaches offer powerful insights into PEG10's interactions, modifications, and regulation:

• Interaction Partners: Immunoprecipitation followed by mass spectrometry (IP-MS) can identify proteins that interact with PEG10 under various conditions. This has revealed interactions with components of the ubiquitination system, suggesting post-translational regulation .

• Post-translational Modifications: Mass spectrometry analysis of purified PEG10 can identify specific modifications such as phosphorylation, ubiquitination, or SUMOylation that may regulate its activity, stability, or localization.

• Protease Substrates: To identify potential substrates of PEG10 protease beyond self-cleavage, comparative proteomic analysis of cells expressing wild-type versus protease-dead mutants can reveal proteins that are differentially processed.

• Structural Proteomics: Techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) or limited proteolysis can provide insights into PEG10's structural dynamics, particularly how frameshift and protease activity affect protein conformation.

• Cross-linking Mass Spectrometry: This approach can map specific interaction interfaces between PEG10 and its binding partners, including TGF-beta receptor ALK1 , providing molecular details of signaling pathway regulation.
  Understanding these proteomic aspects of PEG10 biology is essential for developing targeted interventions in diseases where PEG10 dysfunction contributes to pathology, such as cancer, where it has been implicated in promoting cell growth and inhibiting apoptosis .