Sirt1, the mammalian ortholog of the yeast Sir2 (silent information regulator 2), was shown to play an important role in metabolism and in age-associated diseases, but its role in skeletal homeostasis and osteoporosis has yet not been studied. Using 129/Sv mice with a germline mutation in the Sirt1 gene, we demonstrate that Sirt1 haplo-insufficient (Sirt1(+/-)) female mice exhibit a significant reduction in bone mass characterized by decreased bone formation and increased marrow adipogenesis. Importantly, we identify Sost, encoding for sclerostin, a critical inhibitor of bone formation, as a novel target of Sirt1. Using chromatin immunoprecipitation analysis, we reveal that Sirt1 directly and negatively regulates Sost gene expression by deacetylating histone 3 at lysine 9 at the Sost promoter. Sost down-regulation by small interfering RNA and the administration of a sclerostin-neutralizing antibody restore gene expression of osteocalcin and bone sialoprotein as well as mineralized nodule formation in Sirt1(+/-) marrow-derived mesenchymal stem cells induced to osteogenesis. These findings reveal a novel role for Sirt1 in bone as a regulator of bone mass and a repressor of sclerostin, and have potential implications suggesting that Sirt1 is a target for promoting bone formation as an anabolic approach for treatment of osteoporosis.
Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.
Non-muscle myosin IIA (NMII-A) and the tumor suppressor lethal giant larvae 1 (Lgl1) play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. Here we show that phosphorylation of mammalian Lgl1 by aPKCf prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition of NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. We further show that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6a-aPKCf, and that the formation of these complexes is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6a-aPKCf resides in the leading edge of the cell. Finally, we show that aPKCf and NMII-A compete to bind directly to Lgl1 at the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6a and aPKCf in polarized migrating cells.
Estrogen deficiency leads to rapid bone loss and skeletal fragility. Sclerostin, encoded by the sost gene, and a product of the osteocyte, is a negative regulator of bone formation. Blocking sclerostin increases bone mass and strength in animals and humans. Sirtuin1 (Sirt1), a player in aging and metabolism, regulates bone mass and inhibits sost expression by deacetylating histone 3 at its promoter. We asked whether a Sirt1-activating compound could rescue ovariectomy (OVX)-induced bone loss and biomechanical deterioration in 9-week-old C57BL/6 mice. OVX resulted in a substantial decrease in skeletal Sirt1 expression accompanied by an increase in sclerostin. Oral administration of SRT3025, a Sirt1 activator, at 50 and 100 mg/kg·d for 6 weeks starting 6 weeks after OVX fully reversed the deleterious effects of OVX on vertebral bone mass, microarchitecture, and femoral biomechanical properties. Treatment with SRT3025 decreased bone sclerostin expression and increased cortical periosteal mineralizing surface and serum propeptide of type I procollagen, a bone formation marker. In vitro, in the murine long bone osteocyte-Y4 osteocyte-like cell line SRT3025 down-regulated sclerostin and inactive β-catenin, whereas a reciprocal effect was observed with EX-527, a Sirt1 inhibitor. Sirt1 activation by Sirt1-activating compounds is a potential novel pathway to down-regulate sclerostin and design anabolic therapies for osteoporosis concurrently ameliorating other metabolic and age-associated conditions.
Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.
The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1’s surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.
gamma-Aminobutyric acid (GABA) transporters (GATs) play an important role in inhibitory neurotransmission by clearing synaptically released GABA and by maintaining low resting levels of GABA in synaptic and extrasynaptic regions. In certain brain regions, vesicular zinc is colocalized and coreleased with glutamate and modulates the behavior of a number of channels, receptors, and transporters. We examined the effect of zinc on expressed GATs (GAT1, GAT2, GAT3, and GAT4) in Xenopus laevis oocytes by using tracer flux and electrophysiological methods. We show that zinc is a potent inhibitor of GAT4 (K(i) of 3 muM). Immunolocalization of GAT4 in the hippocampus revealed dense localization in the CA1 and CA3 regions of the hippocampus, regions which are known to be heavily populated by zinc-containing glutamatergic neurons. The results suggest a physiological role of synaptically released zinc in the hippocampus, because zinc released from hyperactive glutamatergic neurons may simultaneously bring about elevated GABAergic inhibition. Therefore, this mode of zinc function signifies a link between excitatory and inhibitory neurotransmission and may play a neuroprotective role against glutamate-induced excitotoxicity.
Modification of proteins by the ubiquitin-like protein, UFM1, requires activation of UFM1 by the E1-activating enzyme, UBA5. In humans UBA5 possesses two isoforms, each comprising an adenylation domain, but only one containing an N-terminal extension.Currently the role of the N-terminal extension in UFM1 activation is not clear. Here we provide structural and biochemical data on UBA5 N-terminal extension to understand its contribution to UFM1 activation. The crystal structures of the UBA5 long isoform bound to ATP with and without UFM1 show that the N-terminus not only is directly involved in ATP binding, but also affects how the adenylation domain interacts with ATP.Surprisingly, in the presence of the N-terminus, UBA5 no longer retains the 1:2 ratio of ATP to UBA5, but rather this becomes a 1:1 ratio. Accordingly, the N-terminus significantly increases the affinity of ATP to UBA5. Finally, the N-terminus, although not directly involved in the E2 binding, stimulates transfer of UFM1 from UBA5 to the E2, UFC1.
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