Recombinant Drosophila melanogaster TM2 domain-containing protein CG10795 (CG10795)

What is the molecular structure of the CG10795 protein?

CG10795, also known as "biscotti" in research literature, is a TM2 domain-containing protein in Drosophila melanogaster with a full-length mature sequence of 160 amino acids (positions 19-178). The protein contains an N-terminal signal sequence followed by a divergent extracellular region, two transmembrane domains connected by a short intracellular loop, and a short C-terminal extracellular tail . The amino acid sequence is: INVDCNELQMMGQFMCPDPARGQIDPKTQQLAGCTREGRARVWCIAANEINCTETGNATFTREVPCKWTNGYHLDTTLLLSVFLGMFGVDRFYLGYPGIGLLKFCTLGGMFLGQLIDIVLIALQVVGPADGSAYVIPYYGAGIHIVRSDNTTYRLPRDDW . The intracellular loop contains a highly conserved DRF (aspartate-arginine-phenylalanine) motif, which is also found in some G-protein coupled receptors and may mediate conformational changes upon ligand binding .

How does CG10795 relate to other TM2D proteins?

CG10795 is the Drosophila ortholog of human TM2D1 and is one of three TM2 domain-containing proteins encoded in the Drosophila genome, alongside almondex (amx, ortholog of TM2D3) and CG11103/amaretto (ortholog of TM2D2) . All three TM2D proteins share similar domain architecture with two transmembrane domains and a conserved intracellular loop containing the DRF motif. While the extracellular regions diverge between the three proteins and across species, the transmembrane domains and intracellular loop sequences are highly conserved evolutionarily . The three TM2D proteins appear to function together in Drosophila, as evidenced by their shared maternal-effect neurogenic phenotypes .

What are the optimal storage and handling conditions for recombinant CG10795 protein?

Recombinant CG10795 protein should be stored at -20°C to -80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . For working aliquots, storage at 4°C for up to one week is recommended. The lyophilized protein powder should be briefly centrifuged prior to opening to bring contents to the bottom. For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (recommended final concentration: 50%) for long-term storage at -20°C/-80°C . The recombinant protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

What expression systems are most effective for producing recombinant CG10795?

Based on the available data, E. coli has been successfully used as an expression system for producing recombinant CG10795 protein . The recombinant product described in the search results features the full-length mature protein (amino acids 19-178) fused to an N-terminal His tag, which facilitates purification. The expression in E. coli yielded protein with greater than 90% purity as determined by SDS-PAGE . For researchers seeking to produce their own recombinant CG10795, this bacterial expression system appears to be an effective approach, though optimization of expression conditions may be necessary depending on the specific experimental requirements.

What is the role of CG10795 in Notch signaling pathways?

CG10795 (biscotti) functions in the Notch signaling pathway, particularly at the γ-secretase cleavage step . Knockout studies of CG10795 in Drosophila have demonstrated that it shares the same maternal-effect neurogenic phenotype as the other TM2D family members, suggesting a crucial role in Notch-mediated neurogenesis during embryonic development . Overexpression of the conserved region of TM2D proteins, including CG10795, acts as a potent inhibitor of Notch signaling specifically at the γ-secretase cleavage step . This indicates that CG10795 is a positive regulator of Notch signaling under normal conditions, and its absence disrupts proper Notch activation, leading to neurogenic defects characterized by excessive neural differentiation at the expense of epidermal development .

What phenotypes are observed in CG10795 knockout models?

CG10795 (biscotti) knockout Drosophila display severe maternal-effect neurogenic phenotypes, which are indistinguishable from those observed in knockouts of the other TM2D family members (almondex and amaretto) . In these maternal-effect mutations, embryos from homozygous mutant mothers exhibit neurogenic defects regardless of their own genotype. Interestingly, triple knockout flies lacking all three TM2D genes do not exhibit any obvious morphological phenotypes beyond those seen in single gene knockouts, and they share the same maternal-effect neurogenic phenotype as the single nulls . This observation suggests that these three genes function together in a non-redundant manner during Notch-mediated neurogenic development, potentially as components of the same molecular complex or pathway .

How do the functions of CG10795 compare with other TM2D family members?

Functional studies have revealed that CG10795 (biscotti), along with the other two TM2D family members in Drosophila (almondex and CG11103/amaretto), share remarkably similar roles in neurogenic development . All three proteins appear to function in concert to regulate Notch signaling, specifically at the γ-secretase cleavage step . Knockout studies demonstrate that loss of any single TM2D gene results in identical maternal-effect neurogenic phenotypes, and triple knockout animals are not phenotypically worse than single nulls . This suggests that these proteins function together, possibly as components of the same molecular complex or pathway, rather than having redundant or compensatory roles . While almondex (the TM2D3 ortholog) appears to be the most well-studied of the three in terms of its neurological functions, with documented roles in adult neuronal maintenance and lifespan , the high conservation of functional domains suggests that CG10795 likely shares similar molecular mechanisms.

What is the evolutionary conservation of CG10795 across species?

CG10795 belongs to a highly conserved family of TM2 domain-containing proteins found throughout metazoans . The human ortholog of CG10795 is TM2D1, which shares the same core domain structure with two transmembrane domains connected by a conserved intracellular loop containing the DRF motif . While the extracellular regions of TM2D proteins are divergent across species, the transmembrane domains and intracellular loop sequences show remarkable evolutionary conservation . This high degree of conservation in the core functional domains suggests that the fundamental molecular mechanisms of CG10795/TM2D1 are likely preserved across species, from Drosophila to humans, making Drosophila models particularly valuable for understanding the basic functions of these proteins in humans .

How does research on CG10795 contribute to understanding Alzheimer's disease mechanisms?

Research on CG10795 and other TM2D family members provides valuable insights into potential mechanisms of Alzheimer's disease (AD) . The human ortholog of CG10795, TM2D1 (also known as BBP, beta-amyloid binding protein), has been shown to interact with Aβ42, Aβ40, and potentially APP (amyloid precursor protein) in previous studies . These interactions appear to involve the extracellular domain and a portion of the first transmembrane domain of TM2D1 . Additionally, overexpression of TM2D1 in human neuroblastoma cells increased their sensitivity to Aβ-induced cell death, suggesting a potential role in mediating Aβ toxicity . Given that rare variants in TM2D3 (the human ortholog of almondex) are associated with AD, and the three TM2D proteins function together in Drosophila, it is likely that the entire TM2D gene family, including CG10795/TM2D1, may be involved in AD pathogenesis . This functional connection makes CG10795 a valuable research target for understanding the molecular mechanisms underlying Alzheimer's disease.

What experimental approaches are most effective for studying functional redundancy among TM2D proteins?

To effectively study the potential functional redundancy or cooperation among TM2D proteins, several experimental approaches have proven valuable. CRISPR/Cas9-mediated homology directed repair (HDR) has been successfully used to generate single, double, and triple knockouts of all three TM2D genes in Drosophila . This genetic approach allows for precise comparison of phenotypes between different knockout combinations to determine whether the genes have redundant, additive, or synergistic functions . Additionally, transgenic rescue experiments can be employed to test whether expression of one TM2D gene can compensate for the loss of another. For biochemical studies of protein interactions and complex formation, co-immunoprecipitation and proximity labeling approaches could be used to identify whether the three TM2D proteins physically interact or are components of the same complex. To study their roles in Notch signaling specifically, γ-secretase activity assays and Notch reporter systems in the context of various TM2D knockouts or overexpression models can provide mechanistic insights . These complementary approaches can elucidate the molecular basis for the observed functional relationships among TM2D family members.

What are the critical considerations when designing experiments to study CG10795's role in neural development?

When designing experiments to investigate CG10795's role in neural development, researchers should consider several critical factors. First, due to the maternal-effect nature of the neurogenic phenotypes, experimental designs must distinguish between maternal and zygotic effects by generating germline clones or using tissue-specific conditional knockouts . Second, the functional overlap with other TM2D family members necessitates careful consideration of genetic backgrounds and potential compensatory mechanisms . Third, since CG10795 functions in Notch signaling, experiments should include appropriate Notch pathway readouts, such as expression of Notch target genes or the activity of Notch-responsive enhancers . Fourth, temporal aspects are crucial, as developmental roles may differ from functions in mature neurons; thus, inducible expression or knockout systems may be valuable . Finally, considering the potential relevance to Alzheimer's disease, researchers might benefit from integrating models or assays that bridge developmental functions with age-related neurodegeneration, such as longitudinal studies of neuronal function in aging flies with manipulated CG10795 expression .

What are the main challenges in studying CG10795 protein interactions and how can they be overcome?

Studying protein interactions of CG10795 presents several challenges due to its transmembrane nature and potential functional redundancy with other TM2D proteins. The protein contains two transmembrane domains connected by a short intracellular loop, making it difficult to express and purify in its native conformation . To overcome these challenges, researchers can employ several strategies: (1) Use specialized detergents or nanodiscs to maintain membrane protein structure during purification; (2) Employ split-reporter systems like BiFC (Bimolecular Fluorescence Complementation) to detect protein-protein interactions in living cells without disrupting membrane localization; (3) Utilize proximity labeling methods such as BioID or APEX to identify neighboring proteins in their native cellular environment; (4) Consider expressing soluble domains separately for interaction studies while validating findings with full-length protein in cellular contexts; and (5) Implement crosslinking approaches prior to immunoprecipitation to stabilize transient interactions. Additionally, considering the shared phenotypes among TM2D proteins, simultaneous analysis of all family members may provide more comprehensive insights into their interactions and functions .

How can researchers effectively differentiate between direct and indirect effects of CG10795 on Notch signaling?

Differentiating between direct and indirect effects of CG10795 on Notch signaling requires a multi-faceted experimental approach. First, temporal analysis of signaling events following CG10795 manipulation can help establish the sequence of molecular changes. Rapid effects following acute depletion or overexpression of CG10795 (using techniques like degron-tagging or optogenetic control) would suggest more direct mechanisms . Second, proximity-based interaction assays can determine whether CG10795 physically associates with Notch pathway components, particularly γ-secretase complex members, which would support direct regulation . Third, in vitro reconstitution of Notch processing using purified components with and without CG10795 can test direct biochemical effects on γ-secretase activity. Fourth, structure-function analyses using domain swapping or point mutations in the conserved regions (particularly the DRF motif) can identify specific sequences required for Notch regulation . Finally, parallel analysis of all three TM2D proteins and epistasis experiments with known Notch pathway components can help position CG10795 within the signaling hierarchy . This comprehensive approach can distinguish between direct modulation of Notch processing and indirect effects through other cellular mechanisms.