Hollow silica nanospheres (HSN) with low densities, large interior spaces and permeable silica shells are suitable for loading enzymes in the cavity to carry out intracellular biocatalysis. The porous shell can protect the encapsulated enzymes against proteolysis and attenuate immunological response. We developed a microemulsion-templating method for confining horseradish peroxidase (HRP) in the cavity of HSN. This simple one-pot enzyme encapsulation method allows entrapping of the enzyme, which retains high catalytic activity. Compared with HRP supported on solid silica spheres, HRP@HSN with thin porous silica shells displayed better enzyme activity. The small HRP@HSN (∼50 nm in diameter), giving satisfactory catalytic activity, can act as an intracellular catalyst for the oxidation of the prodrug indole-3-acetic acid to produce toxic free radicals for killing cancer cells. We envision this kind of hollow nanosystem could encapsulate multiple enzymes or other synergistic drugs and function as therapeutic nanoreactors.

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This is an author's version published in: http://oatao.univ-toulouse.fr/24260 ABSTRACT: Hydrogels are hydrated networks of flexible polymers with versatile biomedical applications, and their resistance to nonspecific protein adsorption is critical. On the other hand, functionalization with other biomacromolecules would greatly enhance their biotechnological potential. The aim of this research is to prepare low fouling hydrogel polymers for selective protein immobilization. Initially, hydrogels were prepared by controlling the composition ratios of 2-carboxyethyl acrylate (CA) and 2-dimethylaminoethyl methacrylate (DMAEMA) monomers in an N,Nmethylene-bis-acrylamide (NMBA) cross-linked free radical polymerization reaction. This series of hydrogels (C1D9 to C9D1) were then analyzed by X-ray photoelectron spectroscopy (XPS) and dynamic laser scattering to confirm the actual polymer ratios and surface charge. When the composition ratio was set at CA:6 vs DMEAMA:4 (C6D4), the hydrogel showed nearly neutral surface charge and an equivalent reaction ratio of CA vs DMAEMA in the hydrogel. Subsequent analysis showed excellent antifouling properties, low blood cell adhesion, hemocompatibility, and platelet deactivation. Moreover, this hydrogel exhibited pH responsiveness to protein adsorption and was then used to facilitate the immobilization of lipase as an indication of active protein functionalization while still maintaining a low fouling status. In summary, a mixed-charge nonfouling pseudozwitterionic hydrogel could be prepared, and its pH-responsive adsorption holds potential for designing a biocompatible tissue engineering matrix or membrane enzyme reactors.

APEX2, an engineered ascorbate peroxidase for high activity, is a powerful tool for proximity labeling applications. Owing to its lack of disulfides and the calcium‐independent activity, APEX2 can be applied intracellularly for targeted electron microscopy imaging or interactome mapping when fusing to a protein of interest. However, APEX2 fusion is often deleterious to the protein expression, which seriously hampers its wide utility. This problem is especially compelling when APEX2 is fused to structurally delicate proteins, such as multi‐pass membrane proteins. In this study, we found that a cysteine‐free single mutant C32S of APEX2 dramatically improved the expression of fusion proteins in mammalian cells without compromising the enzyme activity. We fused APEX2 and APEX2C32S to four multi‐transmembrane solute carriers (SLCs), SLC1A5, SLC6A5, SLC6A14, and SLC7A1, and compared their expressions in stable HEK293T cell lines. Except the SLC6A5 fusions expressing at decent levels for both APEX2 (70%) and APEX2C32S (73%), other three SLC proteins showed significantly better expression when fusing to APEX2C32S (69 ± 13%) than APEX2 (29 ± 15%). Immunofluorescence and western blot experiments showed correct plasma membrane localization and strong proximity labeling efficiency in all four SLC‐APEX2C32S cells. Enzyme kinetic experiments revealed that APEX2 and APEX2C32S have comparable activities in terms of oxidizing guaiacol. Overall, we believe APEX2C32S is a superior fusion tag to APEX2 for proximity labeling applications, especially when mismatched disulfide bonding or poor expression is a concern.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pancancer cohort and found that the SLC1A5 hi /SLC38A2 lo group did not relate to a poor survival rate, whereas the SLC1A5 lo / SLC38A2 hi group significantly aggravated the lethality. Intriguingly, the SLC1A5 hi /SLC38A2 hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.