TBC1D15 Antibody

What is TBC1D15 and what are its primary cellular functions?

TBC1D15 (TBC1 Domain Family, Member 15) is a protein that primarily functions as a regulator of intracellular trafficking. Research demonstrates that it interacts with Ras-like protein in brain 5A, positioning it as a key component in vesicular transport mechanisms . More recent studies have characterized TBC1D15 as having a bipartite structure: a carboxyl-terminal GTPase-activating protein (GAP) domain that acts on the Rab7 GTPase, and a functionally distinct amino-terminal domain that mediates protein-protein interactions critical for cellular signaling pathways . TBC1D15 has been implicated in vesicle trafficking to the lysosome and in the regulation of autophagy, making it a multifunctional protein at the intersection of several cellular processes .

How does TBC1D15 contribute to cellular homeostasis and what signaling pathways involve this protein?

TBC1D15 maintains cellular homeostasis through multiple mechanisms. It has been identified as a critical regulator of autophagy, with studies demonstrating that TBC1D15 depletion results in increased autophagosome formation (measured by elevated LC3-II levels) . Furthermore, it serves as a metabolic regulator, as cells depleted of TBC1D15 show enhanced respiratory capacity and increased oxygen consumption rates, while TBC1D15 overexpression stimulates glycolytic flux . The protein participates in the Numb-p53 signaling axis, where it disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency . Additionally, TBC1D15 interacts with NOTCH1 signaling pathways, which influence mitochondrial metabolism and tumor-initiating cell behavior .

What is the structural basis for TBC1D15 antibody recognition, and which epitopes are commonly targeted?

TBC1D15 antibodies are typically generated against specific regions of the protein to ensure specificity and functionality in various applications. Commercial antibodies often target epitopes within the amino acid ranges 283-404, 624-674, 1-50, or terminal regions (N-term or C-term) of TBC1D15 . The recombinant protein encompassing sequences within the center region of human TBC1D15 is commonly used as an immunogen for antibody production . This structural approach to antibody development enables researchers to select antibodies that recognize specific domains of TBC1D15, facilitating studies on domain-specific functions, interactions, or modifications.

What are the optimal experimental conditions for using TBC1D15 antibodies in different applications?

For Western blotting applications with TBC1D15 antibodies, optimal results are achieved with freshly prepared lysates using RIPA buffer supplemented with protease inhibitors. Protein samples should be resolved on 8-10% SDS-PAGE gels due to TBC1D15's molecular weight (~83 kDa). For immunofluorescence and immunocytochemistry applications, 4% paraformaldehyde fixation for 15 minutes at room temperature, followed by 0.1% Triton X-100 permeabilization, yields optimal results . When performing immunohistochemistry on paraffin-embedded tissues, antigen retrieval using citrate buffer (pH 6.0) is critical for exposing TBC1D15 epitopes. The antibody typically performs optimally at 1:100-1:500 dilutions depending on the specific application and antibody source .

How can researchers validate TBC1D15 antibody specificity in their experimental systems?

Validating TBC1D15 antibody specificity requires a multi-faceted approach. First, researchers should conduct knockdown or knockout experiments using shRNA or CRISPR-Cas9 targeting TBC1D15 (as performed in studies with sgTBC1D15 232, 260, and 295) , followed by Western blotting to confirm signal reduction. Second, researchers should perform immunoprecipitation experiments followed by mass spectrometry to confirm that the antibody pulls down TBC1D15 and known interacting partners like Numb . Third, expressing recombinant tagged TBC1D15 (e.g., myc-TBC1D15) and demonstrating co-detection with both the tag-specific antibody and the TBC1D15 antibody provides additional validation . Finally, testing the antibody across multiple cell lines with known TBC1D15 expression levels, such as HEK-293A and liver tumor-initiating stem-like cells, helps establish consistent performance profiles.

What methodological approaches are most effective for studying TBC1D15 interactions with binding partners like Numb and p53?

To effectively study TBC1D15 interactions with its binding partners, co-immunoprecipitation experiments have proven highly informative. The methodology used in seminal studies involved:

• Reciprocal immunoprecipitations of endogenous, untagged proteins to confirm interactions between TBC1D15 and Numb in multiple cell lines (PIL-4 hepatoblasts and HeLa cells) .

• In vitro binding competition assays where fixed concentrations of Flag-Numb-3A are incubated with purified, hexahistidine-tagged p53 and myc-TBC1D15 .

• Examining the effects of post-translational modifications, particularly phosphorylation of Numb by aPKCζ, on interaction stability by using phosphomimetic mutants (Flag-Numb-3D) versus non-phosphorylatable mutants (Flag-Numb-3A) .

• Sucrose gradient fractionation followed by immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify high-confidence interacting proteins in their native complexes .

These approaches provide comprehensive insights into the dynamic interactions between TBC1D15 and its binding partners under different cellular conditions.

How does TBC1D15 function as an oncoprotein in tumor-initiating cells?

TBC1D15 functions as an oncoprotein through multiple mechanisms that promote tumor-initiating cell (TIC) self-renewal and tumor growth. Research has revealed that TBC1D15:

• Disengages p53 from Numb through its amino-terminal domain, triggering p53 proteolysis and promoting self-renewal and pluripotency .

• Inhibits NOTCH1 activation in CD133+ TICs, affecting mitochondrial metabolism in a way that favors tumor growth .

• Rescues NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruits NOTCH1 to the mitochondrial outer membrane, promoting the generation and expansion of liver TICs .

• Opposes autophagic activity, with TBC1D15 depletion resulting in increased autophagosome formation and induction of catabolic genes including DRAM, SESN2, SCO2, and TIGAR .

• Alters metabolic profiles, with TBC1D15 expression stimulating glycolytic flux while reducing respiratory capacity - a metabolic shift characteristic of many cancer cells .

In vivo tumor formation titration assays demonstrated that stable expression of myc-TBC1D15 enabled as few as 100 TICs to form palpable tumors, while TBC1D15 depletion impaired tumor formation efficiency .

What experimental evidence links TBC1D15 expression to clinical cancer outcomes?

Clinical studies have established compelling connections between TBC1D15 expression and cancer progression. Immunohistochemical analysis of hepatocellular carcinoma (HCC) specimens revealed a striking increase in TBC1D15 expression compared to matched, adjacent noncancerous tissues . Quantitative scoring showed that 42.8±7.3% of tumor cells were strongly immunopositive for TBC1D15, compared to only 5.2±0.5% of noncancerous control cells (P<0.01) .

A comprehensive meta-analysis of TBC1D15 expression across diverse tumor types detected significant (P<0.05) increases in TBC1D15 expression in several cancers, including those derived from breast, prostate, thyroid, and nasopharyngeal tissues . Additionally, researchers identified a correspondence between the expression of TBC1D15 and NANOG in tumors, supporting TBC1D15's role in promoting TIC-mediated oncogenesis .

In surgically recovered tumor implants derived from TICs expressing myc-TBC1D15, expression levels of pluripotency markers OCT4, SOX2, and NANOG were elevated relative to controls, while the differentiation marker ALBUMIN showed reciprocal expression patterns . This data suggests TBC1D15 maintains cancer stemness characteristics associated with poor clinical outcomes.

How can TBC1D15 antibodies be utilized to investigate mitochondrial dynamics in cancer cells?

TBC1D15 antibodies provide valuable tools for investigating mitochondrial dynamics in cancer cells through several methodologies:

• Immunofluorescence microscopy using TBC1D15 antibodies in conjunction with mitochondrial markers can visualize the recruitment of NOTCH1 to the mitochondrial outer membrane by TBC1D15, a process implicated in cancer cell metabolism and TIC expansion .

• Subcellular fractionation followed by immunoblotting with TBC1D15 antibodies can quantify protein distribution between cytosolic, mitochondrial, and other cellular compartments, revealing alterations in mitochondrial association under different conditions.

• Proximity ligation assays (PLA) combining TBC1D15 antibodies with antibodies against mitochondrial outer membrane proteins can provide in situ evidence of protein interactions at the mitochondria.

• In noncancerous tissues, TBC1D15 immunostaining appears in punctate, vesicular structures consistent with its role in endosomal and autophagosomal vesicle trafficking . Comparing this localization pattern with the distribution in cancer cells can reveal pathological alterations in mitochondrial and vesicular dynamics.

These approaches help elucidate how TBC1D15-mediated changes in mitochondrial function and perinuclear localization contribute to cancer cell metabolism and survival.

How does TBC1D15 regulate the balance between autophagy and mitochondrial function?

TBC1D15 serves as a critical molecular switch in the regulation of autophagy and mitochondrial function. Research demonstrates that TBC1D15 depletion results in increased steady-state levels of autophagosomes, as measured by elevated levels of cleaved, lipidated LC3 (LC3-II) . Conversely, enforced expression of Flag-TBC1D15 or its N-terminal domain (Flag-TBC-N) decreases LC3-II levels, consistent with TBC1D15's role in opposing autophagic activity .

At the molecular level, TBC1D15 depletion induces DRAM, a p53 target gene encoding a lysosomal protein that promotes autophagy, along with catabolic genes and p53 effectors SESN2, SCO2, and TIGAR . Metabolic flux analysis reveals that TBC1D15-depleted cells exhibit enhanced respiratory capacity and increased oxygen consumption rates, while TBC1D15 overexpression decreases these parameters and stimulates basal glycolytic flux .

The interplay between TBC1D15 and autophagy is bidirectional: TBC1D15 not only regulates autophagy but is itself regulated by autophagic degradation, particularly under nutrient deprivation conditions . This creates a feedback loop that dynamically adjusts cellular metabolic status and self-renewal capacity. This regulatory network positions TBC1D15 as a nexus between metabolic sensing, mitochondrial function, and stem cell maintenance.

What experimental contradictions exist in the literature regarding TBC1D15 function, and how might these be reconciled?

The literature contains several noteworthy contradictions regarding TBC1D15 function that require careful consideration:

• Conflicting roles in NOTCH signaling: While one study suggests TBC1D15 inhibits NOTCH1 activation in CD133+ TICs , another indicates TBC1D15 stabilizes NOTCH1 by protecting it from degradation . This apparent contradiction might be reconciled by considering context-specific effects or different activation states of NOTCH1 being measured.

• Subcellular localization discrepancies: TBC1D15 has been observed in punctate, vesicular structures in noncancerous tissues , yet other research emphasizes its mitochondrial outer membrane localization . These differences might reflect cell type-specific distributions or dynamic relocalization under different conditions.

• Transcriptional versus protein-level expression: Meta-analysis detected significant increases in TBC1D15 transcription across multiple cancer types, yet in hepatocellular carcinoma, protein elevation was observed despite "lack of significant transcriptional induction" . This suggests post-transcriptional regulatory mechanisms may predominate in certain contexts.

Reconciling these contradictions requires integrated approaches combining genomic, transcriptomic, and proteomic analyses across diverse experimental systems, with careful attention to cellular context, cancer type, and methodological differences between studies.

What novel therapeutic approaches targeting TBC1D15 are being developed for cancer treatment?

Emerging research indicates several promising therapeutic approaches targeting TBC1D15 for cancer treatment:

• NOTCH-TBC1D15 interaction inhibitors have shown potent therapeutic effects in patient-derived xenograft (PDX) mouse models. These inhibitors normalized the perinuclear localization of mitochondria and promoted cytotoxic effects against tumor-initiating cells through NOTCH-dependent pathways .

• RNA interference approaches targeting TBC1D15 have demonstrated efficacy in experimental models. Depletion of TBC1D15 using shRNA impaired tumor formation efficiency in in vivo studies and increased endogenous p53 levels, sensitizing cells to apoptosis induced by the topoisomerase inhibitor etoposide .

• CRISPR-Cas9 mediated knockout of TBC1D15 (using sgTBC1D15 232, 260, and 295) has been employed in intrahepatic inoculation models to evaluate tumor growth inhibition .

• Targeting the amino-terminal domain of TBC1D15, which is responsible for displacing p53 from Numb and triggering p53 proteolysis, represents a potential domain-specific therapeutic strategy to restore p53 tumor suppressor function .

These therapeutic approaches offer promising avenues for targeting cancer stem cells that are often resistant to conventional treatments, potentially reducing tumor recurrence and improving patient outcomes.

What are the common technical challenges when using TBC1D15 antibodies, and how can they be addressed?

Researchers commonly encounter several technical challenges when working with TBC1D15 antibodies:

• Cross-reactivity issues: While some TBC1D15 antibodies show reactivity across human, mouse, and rat samples , others may exhibit species-specific recognition. Researchers should validate antibodies in their specific model system by testing positive and negative controls and comparing multiple antibodies targeting different epitopes.

• Variable detection sensitivity across applications: An antibody that performs well in Western blotting may not be optimal for immunohistochemistry or immunofluorescence. Researchers should select antibodies purified by antigen-affinity chromatography and validated for their specific application of interest.

• Background signal in immunostaining: TBC1D15's punctate, vesicular distribution pattern can sometimes be difficult to distinguish from background staining. This can be addressed by implementing more stringent blocking procedures (5% BSA or normal serum from the secondary antibody host species), using monoclonal antibodies when available, and including appropriate negative controls.

• Detection of isoforms or post-translationally modified forms: TBC1D15 undergoes polyubiquitination on specific lysine residues (K90 and K103 in human) , which may affect antibody recognition. Using antibodies targeting different regions and comparing their detection patterns can help identify potential modifications.

How should researchers interpret complex TBC1D15 expression patterns in heterogeneous tumor samples?

Interpreting TBC1D15 expression in heterogeneous tumor samples requires sophisticated analysis approaches:

• Apply quantitative scoring systems similar to those used in clinical studies, where the percentage of strongly immunopositive cells (e.g., 42.8±7.3% in tumors vs. 5.2±0.5% in noncancerous controls) provides objective metrics for comparison.

• Perform multi-marker co-localization studies combining TBC1D15 antibodies with stem cell markers (CD133, NANOG, OCT4) and differentiation markers (ALBUMIN for liver tissues) to identify specific cell populations with elevated TBC1D15.

• Use digital pathology and image analysis algorithms to quantify staining intensity, subcellular distribution patterns, and co-localization with other markers across different regions of the tumor.

• Complement tissue staining with single-cell RNA sequencing or spatial transcriptomics to correlate TBC1D15 protein expression with transcriptional programs across different tumor microenvironments.

• Consider the subcellular localization pattern of TBC1D15, as it may differ between cancerous (potentially more diffuse or mitochondrial) and noncancerous tissues (punctate, vesicular structures) , providing additional diagnostic information.

This multi-parameter analysis allows researchers to distinguish between different cell populations within heterogeneous tumors and correlate TBC1D15 expression with specific cancer stem cell phenotypes.