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Bioorthogonal click chemistries enable simultaneous spatial patterning of multiple proteins to probe synergistic protein effects on fibroblast function.

ABSTRACT: Three biorthogonal click reactions, a photoinitiated thiol-yne reaction, an azide-alkyne cycloaddition, and a methyltetrazine-transcyclooctene Diels Alder, were used to independently control the presentation of several bioactive proteins to valvular interstitial cells (VICs) in hydrogel scaffolds. Tethered fibroblast growth factor (FGF-2) was found to suppress myofibroblast activation (from 48 ± 7% to 17 ± 6%) and promote proliferation (from 10 ± 2% to 54 ± 3%) at a concentration of 10 ng/mL. In the presence of the pro-fibrotic cytokine transforming growth factor-beta (TGF-β1), FGF-2 could protect the VIC fibroblast phenotype, even at much higher concentrations of TGF-β1 than that of FGF-2. With respect to the fibrocalcific VIC phenotype, TGF-β1 and bone-morphogenic protein-2 (BMP-2) were found to synergistically promote calcific nodule formation (a five-fold increase in nodules compared to TGF-β1 or BMP-2 alone). Exploiting the orthogonal click reactions, FGF-2, TGF-β1 and BMP-2 combinations were patterned into distinct regions on a hydrogel to control VIC activation and nodule formation. Cellular crosstalk between separate regions of the same scaffold was affected by the size of each region as well as the interfacial area between different regions. Collectively, these results demonstrate the versatility and robustness of a photoinitiated thiol-yne reaction to template pendant functionalities that allow for the bioconjugation of multiple proteins. This approach maintains protein bioactivity, providing an in vitro platform capable of achieving a better understanding of the complex mechanisms involved in tissue fibrosis.

SUBMITTER: Ma H

PROVIDER: S-EPMC7396286 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal new features from which we propose spatially linked platforms of newly synthesised protein processing after nuclear import.

Project description:Rationale : Pretargeted radioimmunotherapy (PRIT) based upon bioorthogonal click chemistry has been investigated for the first time in the context of peritoneal carcinomatosis using a CEA-targeting 35A7 mAb bearing trans-cyclooctene (TCO) moieties and several 177Lu-labeled tetrazine (Tz) radioligands. Starting from three Tz probes containing PEG linkers of varying lengths between the DOTA and Tz groups (i.e. PEGn = 3, 7, or 11, respectively, for Tz-1, Tz-2, and Tz-3), we selected [177Lu]Lu-Tz-2 as the most appropriate for pretargeted SPECT imaging and demonstrated its efficacy in tumor growth control. Methods: An orthotopic model of peritoneal carcinomatosis (PC) was obtained following the intraperitoneal (i.p.) injection of A431-CEA-Luc cells in nude mice. Tumor growth was assessed using bioluminescence imaging. Anti-CEA 35A7 mAb was grafted with 2-3 TCO per immunoglobulin. Pretargeted SPECT imaging and biodistribution experiments were performed to quantify the activity concentrations of [177Lu]Lu-Tz-1-3 in tumors and non-target organs to determine the optimal Tz probe for the PRIT of PC. Results: The pharmacokinetic profiles of [177Lu]Lu-Tz-1-3 alone were determined using both SPECT imaging and biodistribution experiments. These data revealed that [177Lu]Lu-Tz-1 was cleared via both the renal and hepatic systems, while [177Lu]Lu-Tz-2 and [177Lu]Lu-Tz-3 were predominantly excreted via the renal system. In addition, these results illuminated that the longer the PEG linker, the more rapidly the Tz radioligand was cleared from the peritoneal cavity. The absorbed radiation dose corresponding to pretargeting with 35A7-TCO followed 24 h later by [177Lu]Lu-Tz-1-4 was higher for tumors following the administration of [177Lu]Lu-Tz-2 (i.e. 0.59 Gy/MBq) compared to either [177Lu]Lu-Tz-1 (i.e. 0.25 Gy/MBq) and [177Lu]Lu-Tz-3 (i.e. 0.18 Gy/MBq). In a longitudinal PRIT study, we showed that the i.p. injection of 40 MBq of [177Lu]Lu-Tz-2 24 hours after the systemic administration of 35A7-TCO significantly slowed tumor growth compared to control mice receiving only saline or 40 MBq of [177Lu]Lu-Tz-2 alone. Ex vivo measurement of the peritoneal carcinomatosis index (PCI) confirmed that PRIT significantly reduced tumor growth (PCI = 15.5 ± 2.3 after PRIT vs 30.0 ± 2.3 and 30.8 ± 1.4 for the NaCl and [177Lu]Lu-Tz-2 alone groups, respectively). Conclusion : Our results clearly demonstrate the impact of the length of PEG linkers upon the biodistribution profiles of 177Lu-labeled Tz radioligands. Furthermore, we demonstrated for the first time the possibility of using bioorthogonal chemistry for both the pretargeted SPECT and PRIT of peritoneal carcinomatosis.

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Bioorthogonal click chemistries enable simultaneous spatial patterning of multiple proteins to probe synergistic protein effects on fibroblast function.

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