title: The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.
abstract: HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes.
authors: Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden.
authors: James
authors: J
authors: Ho
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors: Hendrik
authors: H
authors: Sielaff
authors: Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden.
authors: Aftab
authors: A
authors: Nadeem
authors: Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden. Electronic address: catharina.svanborg@med.lu.se.
authors: Catharina
authors: C
authors: Svanborg
authors: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore. Electronic address: ggrueber@ntu.edu.sg.
authors: Gerhard
authors: G
authors: Grüber
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D006801:Humans; D007768:Lactalbumin / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D058990:Molecular Targeted Therapy; D009829:Oleic Acids / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: C506422:HAMLET complex, human; D009829:Oleic Acids; D000255:Adenosine Triphosphate; D007768:Lactalbumin; D025261:Mitochondrial Proton-Translocating ATPases
keywords: ATP synthase; HAMLET; bioenergetics; molecular motor; tumoricidal protein