Refine
Year of publication
- 2022 (3) (remove)
Document Type
- Article (3)
Language
- English (3)
Has Fulltext
- yes (3)
Is part of the Bibliography
- no (3)
Keywords
- crystal structure (2)
- pancreas (2)
- pancreatitis (2)
- serine protease (2)
- Kazal inhibitor (1)
- N34S (1)
- R122H (1)
- autoproteolysis (1)
- catalytic triad (1)
- isothermal titration calorimetry (ITC) (1)
Institute
Publisher
- Dove Medical Press (1)
- MDPI (1)
- Nature Publishing Group (1)
Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.
Objective: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). This ratio is reversed during chronic alcohol abuse, pancreatic cancer, or pancreatitis due to selectively upregulated expression of TRY2, causing anionic trypsin to become the predominant isoform. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity.
Methods: Trypsin isoforms were recombinantly expressed in E. coli, purified and refolded. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1–TRY1 model.
Results: All trypsin isoforms display similar kinetic properties. The crystal structure of TRY2 reveals that the enzyme crystallized in the autoproteolytic state with Arg122 placed in the S1 binding pocket and the corresponding loop cleaved. The TRY2–TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface.
Conclusion: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated.
(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the SPINK1 gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in E. coli, and inhibitory activities were determined. Crystals of SPINK1–TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.