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Master's Thesis

A study to investigate if Bcl2L1, Bax and BH3-only from Octopus bimaculoides retain the evolutionarily conserved antagonistic tripartite relationship in intrinsic apoptosis.

The Scientific Question

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Apoptosis is a fundamental regulatory process for organism development and tissue homeostasis, and its dysregulation is evident in many diseases.   Intrinsic apoptosis is arbitrated by the B-cell lymphoma 2 Bcl2 family proteins. Unfortunately, the Bcl2 family interaction network is highly complex with multiple redundancies.  Yet their net interactions determine whether a cell lives or dies.  

One way to unravel this entangled relationship is to examine the evolution and emergence of the Bcl-2 family.  Bcl-2 genes emerged early in metazoan evolution and are conserved across their lineage.  Bcl2 family proteins were characterized experimentally on basal metazoans Trichoplax adhaerens, sponge, hydra, and Protostomia-ecdysozoa Drosophila melanogaster  and nematode C. elegans.  The other major clade of Protostomia, Lophotrochozoa, remained uninvestigated. 

 

This project explored whether Bcl-xL, Bax, and BH3-only analogs from Octopus bimaculoides (Lophotrochozoa: Mollusca) exhibit conserved antagonistic relationships similar to those of human apoptosis proteins.

Hypothesis

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Intrinsic apoptosis in O. bimaculoides operates via a tripartite, not bilateral, interaction between Bcl2L1, Bax, and BH3-only proteins.

Research Approach

Wet-Lab Track
Protein Biochemistry and Experimental Validation
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Key technical areas:
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Protein expression, purification, and biochemical validation

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Agarose electrophoresis, SDS-PAGE, Western blot, IMAC, GST purification, SEC

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Pull-down assays to evaluate protein–protein interactions

  • Designed, cloned and optimized expression of OB Bcl-xL, Bax, and BH3-only analogs

  • Validated plasmid cloning with DNA extraction, restriction enzyme digestion, agarose gel electrophoresis, primer design and PCR

  • Purified all proteins via IMAC, GST purification and SEC (~95% purity)

  • Ran SDS-PAGE and Western blot for quality checks

  • Performed binding assays to validate hypothesized interactions

  • Troubleshot expression and purification issues under limited lab conditions

Research Approach

Bioinformatics & Structural Track 
Computational Analysis
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Key technical areas:
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Sequence, motif, MSA, pairwise analyses 

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Phylogenetic analysis

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Structural bioinformatics 

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Functional/binding analyses

  • Conducted MSA, BLAST and domain identification (InterProScan and Consurf)

  • Built phylogenetic trees (MEGA X) to trace evolutionary origins

  • Generated AlphaFold2 structure predictions

  • Compared 3D models via ChimeraX superimposition

  • Identified 12 potential conserved residues mediating antagonistic interactions

  • Mapped predicted interaction networks with STRING

Key Findings

Evolutionary Conservation
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Sequence, structural and phylogenetic analysis suggests OB Bcl2 proteins to be highly conserved, containing the signature BH motifs.  

Evolutionary Conservation

OB BH3-only closely related to human BH3-only Bad

Conserved Interaction Residues 

Validated Protein–Protein Interactions

Why This Matters?

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This project deepens our understanding of how apoptotic mechanisms evolved and highlights shared molecular features across distantly related species.

 

One striking finding was the exceptionally high similarity between the Octopus bimaculoides BH3-only protein and human BH3-only Bad (73% identity; 100% similarity across the aligned region). Structural superimposition further revealed that OB BH3-only most closely resembles trBak, a Bcl2-family protein from Trichoplax adhaerens. Professor Marc’s earlier research proposed that modern BH3-only proteins may have evolved from trBak, and that BH3-only Bad emerged near the dawn of the bilaterian era.

 

Taken together, these observations support an intriguing new idea: the OB BH3-only protein may represent an ancient ancestral form—or early ortholog—of the mammalian BH3-only Bad protein. This perspective offers a fresh angle on how BH3-only proteins diversified from primitive Bak-like ancestors.

 

Beyond evolutionary insight, Octopus bimaculoides itself is an emerging research model due to its remarkable regenerative capacity and sophisticated nervous system. Apoptosis plays a pivotal role in octopus arm regeneration, yet the octopus Bcl2 family remains poorly characterized. With only five putative Bcl2-family proteins identified—compared to the highly expanded human repertoire—octopus provides a simpler, more tractable system for dissecting Bcl2-family interactions. This positions Octopus bimaculoides as a promising model for elucidating individual Bcl2-family roles and for developing future disease-relevant apoptotic models.

Research Project Outputs

Thesis

This thesis investigates the evolution, structure, and functional interactions of Bcl-2 family proteins in Octopus bimaculoides, integrating bioinformatics with wet-lab protein biochemistry to explore how intrinsic apoptosis may have emerged across early metazoans. It presents computational and experimental findings supported by a literature review, hypothesis development, experimental methods, results, and future directions.

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Thesis Presentation

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