Osteoclasts degrade mineralized bone matrix via releasing their lysosomes. However, the mechanisms underlying how lysosomal secretion is regulated in osteoclasts remain largely unknown. PLEKHM1 is a newly identified, causative gene of osteopetrosis in humans and incisors absent (ia/ia) rats. Plekhm1 deficient human and rat osteoclasts exhibit diminished resorptive activity with accumulation of intracellular lysosomes, suggesting that Plekhm1 is a critical regulator of intracellular lysosome trafficking and secretion in osteoclasts. Plekhm1 interacts with Rab7, a lysosome-associated small GTPase, through its C-terminal Rubicon homolog (RH) domain. Overexpression of full-length, but not RH domain-deleted mutant of Plekhm1 by retroviral transduction, rescues bone resorption defect in Plekhm1-/- osteoclasts. Moreover, RH domain-deleted mutant of Plekhm1 fails to be recruited to lysosomes by Rab7. These data led to the hypothesis that the binding of Plekhm1 RH domain to Rab7 plays an important role in regulating lysosome secretion and bone resorption in osteoclasts. The goals of this grant application are to elucidate the molecular details of Plekhm1 and rab7 interaction by crystallography and functional identification of critica residues on Plekhm1 RH domain mediating Rab7 binding. Therefore, we will pursue a) to identify the binding surface and key amino acids mediating the interaction of Plekhm1 RH domain and Rab7 (Aim1); b) to conduct structural-functional analysis of key amino acids in RH domain of Plekhm1 in lysosomal secretion and bone resorption in osteoclasts (Aim2).
Aberrantly enhanced osteoclast activity causes bone loss and fractures, leading to major public health problems. We have identified two proteins in osteoclasts which work together to control osteoclast activity. We plan to determine how these proteins interact in osteoclasts, raising the possibility that we will be able to provide new therapeutic targets for the treatment of bone loss in metabolic bone diseases such as osteoporosis and arthritis.
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