Défense publique de thèse - Louise GÉRARD

Investigation of the ABCB5β transporter dimerization status

ABCB5 belongs to the ABC transporter superfamily composed of 48 members. These transporters have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma stem cells, and limbal stem cells. Its expression has also been reported, among other, in melanoma, colorectal cancer, hepatocellular carcinoma and several hematological malignancies. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half-transporter (ABCB5β). Several studies have shown that the ABCB5β homodimer, in contrast to ABCB5FL, cannot confer multidrug resistance. Since these studies focused on a limited number of drugs, we cannot exclude the possibility that this homodimer may be involved in drug resistance or biological functions that have yet to be elucidated. However, it is also reasonable to hypothesize that ABCB5β could dimerize with other half transporters of the ABCB family to become functional. Using three complementary techniques: (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in two melanoma cell lines: ABCB5β/B6 and ABCB5β/B9. Both heterodimers could be expressed in High-Five insect cells when both interacting partners were fused using the linker region of ABCB1, resulting in chimeric heterodimers. ATPase assays revealed that all chimeric heterodimers have a basal ATPase activity and that both functional nucleotide-binding domains in each dimer are required for their basal ATPase activity. Also, we obtained preliminary data suggesting that ABCB5β traffics from the endoplasmic reticulum to melanosomes independently of its dimerization, suggesting that its heterodimers might be located in similar organelles. However, several limitations were encountered when attempting to confirm their intracellular localization. Finally, since several anti-ABCB5 antibodies in the literature have shown a lack of specificity, we generated a mouse monoclonal anti-ABCB5 antibody in collaboration with Atlas Antibodies. The specificity of this antibody was demonstrated by immunofluorescence, making this antibody an important tool in the characterization of ABCB5β and ABCB5FL. Although further studies are needed to elucidate the physiological relevance of ABCB5β heterodimers, preliminary data support the hypothesis that ABCB5β is involved in melanogenesis. Taken together, these results represent an important step towards elucidating the functional role of ABCB5β in melanocytes and melanoma.

Contact : Louise GÉRARD - 081724263 - louise.gerard@unamur.be
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