title: TRAFD1 (FLN29) Interacts with Plekhm1 and Regulates Osteoclast Acidification and Resorption.
abstract: Plekhm1 is a large, multi-modular, adapter protein implicated in osteoclast vesicle trafficking and bone resorption. In patients, inactivating mutations cause osteopetrosis, and gain-of-function mutations cause osteopenia. Investigations of potential Plekhm1 interaction partners by mass spectrometry identified TRAFD1 (FLN29), a protein previously shown to suppress toll-like receptor signaling in monocytes/macrophages, thereby dampening inflammatory responses to innate immunity. We mapped the binding domains to the TRAFD1 zinc finger (aa 37-60), and to the region of Plekhm1 between its second pleckstrin homology domain and its C1 domain (aa 784-986). RANKL slightly increased TRAFD1 levels, particularly in primary osteoclasts, and the co-localization of TRAFD1 with Plekhm1 also increased with RANKL treatment. Stable knockdown of TRAFD1 in RAW 264.7 cells inhibited resorption activity proportionally to the degree of knockdown, and inhibited acidification. The lack of acidification occurred despite the presence of osteoclast acidification factors including carbonic anhydrase II, a3-V-ATPase, and the ClC7 chloride channel. Secretion of TRAP and cathepsin K were also markedly inhibited in knockdown cells. Truncated Plekhm1 in ia/ia osteopetrotic rat cells prevented vesicle localization of Plekhm1 and TRAFD1. We conclude that TRAFD1, in association with Plekhm1/Rab7-positive late endosomes-early lysosomes, has a previously unknown role in vesicle trafficking, acidification, and resorption in osteoclasts.
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655 United States of America.
authors.forename: Hanna
authors.initials: H
authors.lastname: Witwicka
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655 United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Jia
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655 United States of America.
authors.forename: Artem
authors.initials: A
authors.lastname: Kutikov
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655 United States of America.
authors.forename: Pablo
authors.initials: P
authors.lastname: Reyes-Gutierrez
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655 United States of America.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655 United States of America.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Odgren
mesh_terms: D000143:Acids / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D000071183:Autophagy-Related Proteins; D002454:Cell Differentiation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
chemical_list: D000143:Acids; D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; C534487:FLN29 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C476215:PLEKHM1 protein, human; D012333:RNA, Messenger