Recombinant Mouse Pleckstrin homology domain-containing family M member 3 (Plekhm3)

What is the basic structure and function of Plekhm3?

Plekhm3 (Pleckstrin Homology Domain Containing, Family M, Member 3) is a protein containing pleckstrin homology domains, which are typically involved in intracellular signaling and membrane trafficking . The mouse Plekhm3 protein consists of 761 amino acids and includes several functional domains that mediate interactions with cellular components .

The protein plays a critical role in cellular processes including autophagy and intracellular trafficking . It appears to function through interactions with Rab GTPases and various cargoes, mediating lysosomal biogenesis and degradation processes . The modulation of these pathways is essential for maintaining cellular homeostasis and responding to stress and metabolic conditions .

How is Plekhm3 expressed in normal versus pathological conditions?

Studies have shown differential expression of Plekhm3-derived circular RNA (circ-PLEKHM3) between normal and cancer tissues. Specifically, circ-PLEKHM3 was found to be significantly downregulated in ovarian cancer tissues compared to normal tissues . The expression level was also markedly reduced in ovarian cancer cell lines (SKOV3 and A2780) compared to normal ovarian epithelial cells (IOSE-80) .

This downregulation suggests that reduced Plekhm3 expression may contribute to cancer progression, while its restoration could potentially inhibit tumor growth. This is supported by experimental evidence showing that overexpression of circ-PLEKHM3 reduced colony-formation ability and promoted apoptosis in ovarian cancer cells .

What are the available research tools for studying Plekhm3?

Several research tools have been developed to facilitate the study of Plekhm3:

• Recombinant Proteins: Recombinant Mouse Plekhm3 protein (AA 1-761) with His tag, expressed in HEK-293 cells, is available for biochemical and structural studies .

• Knockout Cell Lines: PLEKHM3 knockout cell lines (e.g., HEK293) have been developed using techniques such as electric rotation method or viral method . These cells have been confirmed negative for mycoplasma and are maintained in 90% DMEM + 10% FBS .

• Expression Vectors: Circ-PLEKHM3 overexpression vectors (oe-circ-PLEKHM3) have been constructed and used in transfection experiments to study the effects of Plekhm3 upregulation .

• Cell Culture Models: Various cell lines, including SKOV3 and A2780 ovarian cancer cells, have been used to study Plekhm3 function in vitro .

How does circ-PLEKHM3 interact with miRNA networks to regulate gene expression?

Circular RNAs often function as microRNA (miRNA) sponges, regulating gene expression by sequestering specific miRNAs. Research has revealed that circ-PLEKHM3 targets and sponges miR-320a, which is upregulated in ovarian cancer tissues and cells . The expression level of miR-320a in ovarian cancer tissues was found to be negatively correlated with circ-PLEKHM3 expression .

This interaction was confirmed through dual-luciferase reporter assays, where miR-320a overexpression induced a 65% reduction in luciferase activity in the circ-PLEKHM3 wild-type group, but showed little influence on the circ-PLEKHM3 mutant group . RNA immunoprecipitation (RIP) analysis further demonstrated that both circ-PLEKHM3 and miR-320a were enriched in anti-Ago2 immunoprecipitates, confirming their physical interaction in the RNA-induced silencing complex .

Through this sponging mechanism, circ-PLEKHM3 indirectly regulates the expression of SMG1, a target gene of miR-320a, thereby influencing cancer cell proliferation and apoptosis .

What are the molecular mechanisms underlying curcumin's effect on Plekhm3 expression?

Curcumin has been shown to significantly upregulate circ-PLEKHM3 abundance in ovarian cancer cells in a dose-dependent manner . While the precise molecular mechanisms remain under investigation, experimental data provides insights into this regulatory pathway:

• Exposure to increasing concentrations of curcumin (10, 20, or 40 μM) progressively elevated circ-PLEKHM3 levels in SKOV3 and A2780 cells .

• Concurrently, curcumin treatment reduced miR-320a abundance in a dose-dependent pattern .

• This dual regulation creates a favorable environment for circ-PLEKHM3 to exert its tumor-suppressive functions.

The reciprocal relationship between curcumin treatment, circ-PLEKHM3 upregulation, and miR-320a downregulation suggests that curcumin may inhibit ovarian cancer progression through modulating this regulatory axis . Further research is needed to elucidate the direct molecular targets of curcumin that initiate this cascade.

How does Plekhm3 disruption affect autophagy and lysosomal function at the molecular level?

PLEKHM3 knockout models reveal that this protein plays essential roles in autophagy and intracellular trafficking pathways . The absence of PLEKHM3 alters autophagic flux and impacts lysosomal function, providing a unique opportunity to explore mechanisms related to lysosomal storage disorders and neurodegeneration .

At the molecular level, Plekhm3 operates through interactions with Rab GTPases and various cellular cargoes, mediating lysosomal biogenesis and degradation processes . The disruption of these interactions in knockout models leads to dysregulated autophagy, potentially affecting:

• Cargo selection and sequestration in autophagosome formation

• Autophagosome-lysosome fusion efficiency

• Degradation of cellular components within autolysosomes

• Recycling of macromolecules back to the cytoplasm

These alterations collectively contribute to disturbed cellular homeostasis, potentially explaining the association between Plekhm3 dysfunction and disease states .

What are the optimal conditions for expressing and purifying recombinant Plekhm3 protein?

Based on successful expression protocols, the following conditions are recommended for expressing and purifying recombinant mouse Plekhm3 protein:

• Expression System: Human embryonic kidney (HEK-293) cells have proven effective for expressing recombinant mouse Plekhm3 . This mammalian expression system ensures proper folding and post-translational modifications of the protein.

• Protein Design: The full-length protein (AA 1-761) with a His tag facilitates one-step affinity chromatography purification .

• Vector Design: State-of-the-art algorithms for plasmid design and gene synthesis optimize expression efficiency .

• Purification Method: One-step affinity chromatography using the His tag is sufficient for obtaining pure protein .

• Quality Control: Verification of protein identity and purity through SDS-PAGE and Western blotting is essential before using the recombinant protein in downstream applications.

This approach ensures reliability for producing intracellular, secreted, and transmembrane proteins with high yield and biological activity .

How can researchers effectively manipulate Plekhm3 expression in experimental models?

Several approaches have been employed to manipulate Plekhm3 expression in experimental models:

• Overexpression Systems:

  • Circ-PLEKHM3 overexpression vectors (oe-circ-PLEKHM3) can be constructed and transfected into cells using Lipofectamine 3000 .

  • Typical transfection protocols involve using 4.0 μg of constructed vectors for optimal expression .

• Knockout Models:

  • PLEKHM3 knockout cell lines can be generated using electric rotation method or viral method .

  • These knockout cells are typically cultured in 90% DMEM + 10% FBS and maintained with appropriate passage ratios (1:3~1:6) .

• Pharmacological Modulation:

  • Curcumin treatment (10-40 μM) for 24-72 hours can be used to upregulate circ-PLEKHM3 expression in cancer cell lines .

  • Dose-dependent and time-dependent effects should be carefully monitored for optimal results .

• RNA Interference:

  • MiR-320a mimic or inhibitor can be used to modulate the circ-PLEKHM3/miR-320a/SMG1 axis .

  • Typical transfection protocols involve using 50 nM of oligos with Lipofectamine 3000 .

What assays are most effective for evaluating Plekhm3 function in cellular processes?

Based on published research methodologies, the following assays have proven effective for evaluating Plekhm3 function:

• Cell Proliferation Assays:

  • Cell Counting Kit-8 (CCK-8) assay can measure cell viability by detecting the absorbance at 450 nm after incubation with CCK-8 reagent .

  • Protocol: Plate 1 × 10^4 cells per well, treat as required, add 10 μL CCK-8 reagent, incubate for 3 hours at 37°C, and measure absorbance .

• Colony Formation Assay:

  • This assay evaluates long-term proliferative capacity by quantifying visible colonies after extended culture periods .

  • Protocol: Following treatment, culture cells for 12 days, fix with 4% paraformaldehyde, stain with 0.5% crystal violet, and count visible colonies .

• Apoptosis Assessment:

  • Western blotting for apoptotic markers (c-caspase-3, Bax) and proliferation markers (PCNA) provides insights into cell death and survival pathways .

• Molecular Interaction Assays:

  • Dual-luciferase reporter assay can confirm direct interactions between circ-PLEKHM3 and miRNAs .

  • RNA immunoprecipitation (RIP) assay using Magna RIP kit can verify RNA-protein interactions within the RNA-induced silencing complex .

How should researchers analyze and interpret contradictory findings in Plekhm3 research?

When faced with contradictory findings in Plekhm3 research, researchers should:

• Examine Experimental Conditions:

  • Different cell types or tissues may express Plekhm3 differently or respond differently to its manipulation.

  • Variations in experimental conditions (e.g., curcumin concentration, treatment duration) can significantly impact results .

• Consider Regulatory Networks:

  • Plekhm3 functions within complex regulatory networks involving miRNAs and target genes .

  • Contradictory findings might reflect different components of these networks being affected in different experimental systems.

• Validate with Multiple Approaches:

  • Use complementary experimental approaches to verify findings.

  • For example, combine overexpression and knockout studies, or use both in vitro and in vivo models.

• Statistical Analysis:

  • Apply appropriate statistical tests (e.g., Student's t-test for comparing two groups, ANOVA with Tukey's post hoc test for multiple groups) .

  • Ensure experiments are conducted with sufficient replicates (minimum three independent experiments) .

• Consider Context-Dependency:

  • Plekhm3 function may be context-dependent, varying based on cellular state, disease progression, or microenvironment.

What criteria should be used to assess the quality and relevance of recombinant Plekhm3 for specific applications?

Researchers should evaluate recombinant Plekhm3 based on the following criteria:

• Expression System Compatibility:

  • Proteins expressed in mammalian cells (e.g., HEK-293) typically maintain proper folding and post-translational modifications .

  • This is particularly important for proteins involved in complex cellular processes like Plekhm3.

• Protein Integrity:

  • Full-length proteins (AA 1-761) may be necessary for studies requiring complete functional domains .

  • For domain-specific studies, partial proteins may be more appropriate.

• Tag Selection:

  • His-tagged proteins facilitate purification but may affect protein function in some applications .

  • The impact of tags on protein function should be assessed experimentally.

• Purity and Yield:

  • Proteins purified by one-step affinity chromatography should meet purity standards suitable for the intended application .

  • Additional purification steps may be necessary for highly sensitive assays.

• Biological Activity:

  • Functional assays should confirm that the recombinant protein retains its expected biological activities.

  • This is particularly important for proteins intended for interaction studies or enzymatic assays.

How can researchers integrate findings from Plekhm3 studies into broader understanding of cellular pathways?

To integrate Plekhm3 findings into broader cellular pathway understanding:

• Pathway Mapping:

  • Place Plekhm3 within established cellular pathways such as autophagy, lysosomal function, and intracellular trafficking .

  • Identify points of convergence with other regulatory pathways, such as miRNA networks .

• Multi-omics Integration:

  • Combine findings from genomics, transcriptomics, proteomics, and metabolomics studies to create a comprehensive view of Plekhm3 function.

  • This approach may reveal unexpected connections between Plekhm3 and other cellular processes.

• Disease Relevance Assessment:

  • Connect Plekhm3 dysfunction to specific disease mechanisms, such as cancer progression or lysosomal storage disorders .

  • This translational approach enhances the clinical relevance of basic research findings.

• Comparative Analysis:

  • Compare Plekhm3 function across different species, cell types, and disease states to identify conserved mechanisms.

  • Evolutionary conservation often indicates fundamental biological importance.

• Computational Modeling:

  • Develop computational models that incorporate Plekhm3 within cellular pathway networks.

  • These models can generate testable hypotheses and predict outcomes of experimental manipulations.