Allosteric Activation of Transglutaminase 2 via Inducing an “Open” Conformation for Osteoblast Differentiation

Abstract Osteoblasts play an important role in the regulation of bone homeostasis throughout life. Thus, the damage of osteoblasts can lead to serious skeletal diseases, highlighting the urgent need for novel pharmacological targets. This study introduces chemical genetics strategy by using small molecule forskolin (FSK) as a probe to explore the druggable targets for osteoporosis. Here, this work reveals that transglutaminase 2 (TGM2) served as a major cellular target of FSK to obviously induce osteoblast differentiation. Then, this work identifies a previously undisclosed allosteric site in the catalytic core of TGM2. In particular, FSK formed multiple hydrogen bonds in a saddle‐like domain to induce an “open” conformation of the β‐sandwich domain in TGM2, thereby promoting the substrate protein crosslinks by incorporating polyamine. Furthermore, this work finds that TGM2 interacted with several mitochondrial homeostasis‐associated proteins to improve mitochondrial dynamics and ATP production for osteoblast differentiation. Finally, this work observes that FSK effectively ameliorated osteoporosis in the ovariectomy mice model. Taken together, these findings show a previously undescribed pharmacological allosteric site on TGM2 for osteoporosis treatment, and also provide an available chemical tool for interrogating TGM2 biology and developing bone anabolic agent.

and the absorbance of the solution was quantificationally measured at 562 nm with a spectrophotometer (BioTek, USA).

ALP activity assay
MC3T3-E1 cells were treated with FSK (0.5, 1, 5 μM) to induce osteogenesis differentiation for 5 days. Then, the cells were lysed with 1% triton in PBS and ALP activity was detected with ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

Immunoblots
MC3T3-E1 cells were lysed in NP-40 Lysis Buffer (Beyotime, Shanghai, China) supplemented 1% protease inhibitor. The concentration of protein was tested with BCA method (TransGen, Beijing, China). Proteins were separated by 10-15% SDS-PAGE and electro-transferred onto PVDF membranes. The membranes were blocked with 5% nonfat-dried milk for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were washed and incubated with secondary antibody conjugated HRP for 2 h at room temperature. Finally, immunoblots were visualized by a Tanon 5200 Imaging Analysis System (Tanon, Shanghai, China).

Click chemistry for target identification
MC3T3-E1 cells were incubated with AD-FSK (50 μM) for 4 h and exposed to 365 nm UV light for 10 min. Cells were lysed in NP-40 Lysis Buffer (Beyotime, Shanghai, China) supplemented 1% protease inhibitor and adjusted to a consistent concentration and volume with NP-40 Lysis Buffer. Cells lysates containing 1 mg proteins were successively mixed with 3 μL Biotin-PEG 3 -N 3 (20 mM), 11.6 μL TCEP (14.4 mg/mL), and 1.75 μL TBTA (34 mM). The click reaction was initiated by 11.6 μL CuSO 4 (50 mM). The reaction was incubated for 1 h at room temperature. The protein pellets were collected by 6000 g centrifugation and were washed with cold methanol twice. Then, the samples were heated in 1.2% SDS at 85 °C for 5 5 min. Then streptavidin beads (30 μL) were mixed with samples and incubated at room temperature for 3 h. The biotinylated proteins captured on streptavidin beads were digested to peptides with trypsin. These peptides were analyzed by LC-MS/MS (NanoLC-LTQ Velos pro MS, Thermo, Waltham, MA, USA).
These peptides were separated in C18 reversed-phase column. The elution buffer A was 0.1% formic acid in water and the elution buffer B was 0.1% formic acid in ACN. The procedure for gradient eluting were 2%-40% B for 70 min; 40%-95% B for 5 min; 95%B for 20 min. The eluent was introduced to the MS at 300 nL/min. MS parameters were listed as follows: scan spectra, 350 to 2000 m/z; maximum IT, 50 ms; HCD Collision Energy, 35%; resolution, 60000; Target FDR for PSMs, 0.01. MS data were analyzed with Proteome Discoverer (version 1.4) software with SEQUEST (Thermo, Waltham, MA, USA).

Cellular thermal shift assay (CETSA)
MC3T3-E1 cells were treated with FSK (50 μM) or DMSO for 2 h at 37 °C. Cells were harvested in PBS containing 1% protease inhibitor and divided into ten groups. The samples were heated for 2 min respectively at gradient temperatures (37-55 °C) with T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). 20 μL kinase buffer (CST, Beverly, MA, USA) was added into the samples and repeated freeze-thaw in liquid nitrogen was performed to obtain cell lysates. The cell lysates were analyzed by immunoblot with anti-TGM2 antibody.

TGM2 expression and purification
TGM2 was subcloned into pET-28a vector to create an N-terminal His tag in TGM2.

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Escherichia coli Bl21 (DE3) cells (Solarbio, Beijing, China) were transformed with the vector and monoclonal bacterial colony was inoculated 500 mL LB medium (10 g/L peptone, 10 g/L NaCl and 5 g/L yeast extract). The bacteria were grown at 37 °C in 200 rpm until OD 600 reached 0.6. Then, 1 mM isopropyl β-D-thiogalactoside (IPTG) was added at 16 °C in 120 rpm for 24 h, and the cells were harvested by 4000 rpm centrifugation. Cell pellets were lysed by sonication in 30 mL binding buffer (20 mM HEPES, 250 mM NaCl, 10 mM imidazole). Cell debris was removed by 12000 rpm centrifugation and the supernatant was loaded onto Ni-NTA column (Thermo, Waltham, MA, USA), which was washed by 100 mL binding buffer. After 10 min, the column was washed with 100 mL washing buffer (20 mM HEPES, 250 mM NaCl, 50 mM imidazole) to remove impurities. The TGM2 protein was eluted with elution buffer (20 mM HEPES, 250 mM NaCl, 250 mM imidazole). Proteins were concentrated by centrifugal filtration (Amicon Ultra-15, Millipore, MA, USA) with a molecular weight cutoff of 10 kDa. The protein was loaded onto a molecular sieve chromatography and eluted with PBS. Purity of TGM2 was tested by SDS/PAGE with coomassie brilliant blue staining.

Surface plasmon resonance (SPR) assay
The ability of FSK to bind TGM2 was analyzed by Biacore 8K system (GE Healthcare, Uppsala, Sweden). The recombinant TGM2 protein (50 μg/mL) was immobilized on

Identification of FSK-binding peptides on TGM2
TGM2 protein was diluted with TNC buffer to 300 μg/mL and incubated with FSK (50 μM) or equal DMSO on ice for 1 h. Then, the samples were digested with 5 μg/mL pronase (Solarbio, Beijing, China) for 20 min in 1×TNC buffer at 37 °C, and the reaction was terminated by SDS loading buffer. Finally, SDS-PAGE was conducted to separate the samples to determine degradation product. The degradation protein bands were excised and analyzed with LC-MS/MS.

Identification of FSK-binding sites on TGM2
TGM2 protein (100 μg/mL) was incubated with AD-FSK (50 μM) or equal DMSO for 1 h on ice. Then, the samples were exposed to 365 nm UV light for 10 min on ice. The samples were mixed with SDS loading buffer and heated at 98 °C for 10 min. Subsequently, the samples were separated with SDS-PAGE and the main protein band was excised. The proteins were digested with trypsin and analyzed with LC-MS/MS.

TGM2 knockdown with shTGM2 lentivirus 8
The lentivirus containing shTGM2 was established from Hanbio, Shanghai, China. The lentivirus was transfected into MC3T3-E1 cells with polybrene (8 μg/mL) for 24 h. The cells were screened by the addition of puromycin (10 μg/mL) to establish the stable TGM2 knockdown cells.

TGM2-interaction proteins discovery with APEX2
TGM2-APEX2 plasmid (30 μg Images were obtained by laser confocal microscopy (100×) (LSM880, Zeiss, Oberkochen, Germany) with 490 nm exciting wavelength and 516 nm emission wavelength. The images were quantificationally analyzed by Image J (version 1.48) with MiNA 2.0 functional plug for calculating the mitochondrial mean branch length. Each group was evaluated by 6 original images. Statistical analysis was conducted by T-test.

Hydrogen-deuterium exchange mass spectrometry (HDXMS) analysis
TGM2 protein (1 mg/mL) was incubated with FSK (50 μM) or equivalent DMSO in 4 °C for 30 min. The protein was equilibrated in D 2 O buffer in room temperature and quenched with 37.5% hydrochloric acid at indicated times. Samples were digested by Waters ENZYMATE BEH 9 pepsin column. Then the peptides were captured on a VanGuard Pre-Column trap, eluted with 15% acetonitrile and separated using an ACQUITY UPLC BEH C18 column. Relative deuterium levels of all peptides were calculated by subtracting the mass of undeuterated control sample from that of the deuterium-labeled sample.

FlAsH-based FRET
HEK293T cells were cultured in 6 cm dishes to transfect FRET plasmids with Lipofectamine 2000. 48 hours later, cells were digested and planted in 24-well confocal plates. The 5 μM FSK was treated for 2 hours after adherence. Then cells were washed three times with HBSS and treated with 250 nM FlAsH-EDT2 in 37 °C for 20 min. Whereafter, cells were washed three times with 250 μM EDT in HBSS to remove non-specific adsorption and maintained with clean HBSS. Fluorescence images were scanned with laser confocal microscopy (LSM880, Zeiss, Oberkochen, Germany). ECFP was excited at 405 nm, and FlAsH was excited at 514 nm directly. Emission of ECFP and FlAsH was measured at 480 ± 20 nm and 530 ± 20 nm, respectively. FRET ratio was calculated as the ratio of F530 to F480.
Fluorescence signals were digitalized using Zen software.

Ovariectomy-induced osteoporosis model
Six-week-old C57bl/6 female mice were anesthetized with isoflurane and shaved on dorsum.
A dorsal midline incision was made to exteriorize the ovary. Then the fallopian tube was ligated and the ovary was removed and the wound was sutured. The same process was repeated on the other side. Sham operations were performed in the same way without ligating fallopian tube and removing ovaries. One week after the surgery, the FSK or ALN dissolved in normal saline containing 5 %DMSO and 5% PEG 300 was treated with intraperitoneal injection every other day for 16 weeks.

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Microstructure of bone trabeculae parameters were analyzed with CTAn software.

Bone sectioning and immunohistochemistry analysis
After sacrifice, the femora were fixed in 4% paraformaldehyde at room temperature for 24 h.
Then the femora were decalcified in 10% EDTA (pH=7.5) for 21d. The tissues were dehydrated and embedded in paraffin. Then 4.5 μm-think sections were cut with rotary microtome (RM215, Leica, Germany). The dewaxed sections were eliminated endogenous peroxidase activity and blocked with goat serum. Then sections were incubated in the anti-COX4 and anti-OCN primary antibodies in 4 °C overnight and incubated in secondary antibody for 30 min and visualized with Diaminobenzidine. Sections were scanned with Digital pathology scanner (WS-10, Wisleap, China).