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1.
Cells ; 12(17)2023 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-37681871

RESUMEN

Osteoclasts are multinucleated, bone-resorbing giant cells derived from monocyte-macrophage cell lines. Increased bone resorption results in loss of bone mass and osteoporosis. Osteoclast and bone marrow macrophages have been shown to express three TG enzymes (TG2, Factor XIII-A, and TG1) and TG activity to regulate osteoclast differentiation from bone marrow macrophages in vitro. In vivo and in vitro studies have demonstrated that the deletion of TG2 causes increased osteoclastogenesis and a significant loss of bone mass in mice (Tgm2-/- mice). Here, we confirm that TG2 deficiency results in increased osteoclastogenesis in vitro and show that this increase can be reversed by a TG inhibitor, NC9, suggesting that other TGs are responsible for driving osteoclastogenesis in the absence of TG2. An assessment of total TG activity with 5-(biotinamido)-pentylamine, as well as TG1 and FXIII-A activities using TG-specific Hitomi peptides (bK5 and bF11) in Tgm2-/- bone marrow flushes, bone marrow macrophages, and osteoclasts, showed a significant increase in total TG activity and TG1 activity. Factor XIII-A activity was unchanged. Aspartate proteases, such as cathepsins, are involved in the degradation of organic bone matrix and can be produced by osteoclasts. Moreover, Cathepsin D was shown in previous work to be increased in TG2-null cells and is known to activate TG1. We show that Pepstatin A, an aspartate protease inhibitor, blocks osteoclastogenesis in wild-type and Tgm2-/- cells and decreases TG1 activity in Tgm2-/- osteoclasts. Cathepsin D protein levels were unaltered in Tgm2-/-cells and its activity moderately but significantly increased. Tgm2-/- and Tgm2+/+ bone marrow macrophages and osteoclasts also expressed Cathepsin E, and Renin of the aspartate protease family, suggesting their potential involvement in this process. Our study brings further support to the observation that TGs are significant regulators of osteoclastogenesis and that the absence of TG2 can cause increased activity of other TGs, such as TG1.


Asunto(s)
Proteasas de Ácido Aspártico , Osteoclastos , Animales , Ratones , Osteogénesis , Catepsina D , Transglutaminasas/genética , Ácido Aspártico , Factor XIII
2.
Molecules ; 28(4)2023 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-36838622

RESUMEN

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.


Asunto(s)
Factor XIIIa , Transglutaminasas , Transglutaminasas/química , Factor XIIIa/química , Factor XIIIa/metabolismo , Colorantes Fluorescentes , Técnicas de Cultivo de Célula , Macrófagos/metabolismo
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