Almost all commercial proteins are purified using ammonium sulfate precipitation. Protein-polymer conjugates are synthesized from pure starting materials, and the struggle to separate conjugates from ...polymer, native protein, and from isomers has vexed scientists for decades. We have discovered that covalent polymer attachment has a transformational effect on protein solubility in salt solutions. Here, protein-polymer conjugates with a variety of polymers, grafting densities, and polymer lengths are generated using atom transfer radical polymerization. Charged polymers increase conjugate solubility in ammonium sulfate and completely prevent precipitation even at 100% saturation. Atomistic molecular dynamic simulations show the impact is driven by an anti-polyelectrolyte effect from zwitterionic polymers. Uncharged polymers exhibit polymer length-dependent decreased solubility. The differences in salting-out are then used to simply purify mixtures of conjugates and native proteins into single species. Increasing protein solubility in salt solutions through polymer conjugation could lead to many new applications of protein-polymer conjugates.
Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to ...grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.
The power and elegance of protein-polymer conjugates has solved many vexing problems for society. Rational design of these complex covalent hybrids depends on a deep understanding of how polymer ...physicochemical properties impact the conjugate structure-function-dynamic relationships. We have generated a large family of chymotrypsin-polymer conjugates which differ in polymer length and charge, using grafting-from atom-transfer radical polymerization, to elucidate how the polymers influenced enzyme structure and function at pHs that would unfold and inactivate the enzyme. We also used molecular dynamics simulations to deepen our understanding of protein-polymer intramolecular interactions. Remarkably, the data revealed that, contrary to current thoughts on how polymers stabilize proteins, appropriately designed polymers actually stabilize partially unfolded intermediates and assist in refolding to an active conformation. Long, hydrophilic polymers minimized interfacial interactions in partially unfolded conjugates leading to increased stabilization. The design of covalently attached intramolecular biomimetic chaperones that drive protein refolding could have far reaching consequences.
The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions ...between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.
Immune dysfunction due to microgravity remains a hurdle in the next step of human space exploration. Dendritic cells (DC) represent a critical component of immunity, given their role in the detection ...of invaders and the subsequent task of activating T cells to respond and eliminate the threat. Upon encounter with microbes, DC undergo a process of maturation, whereby the cells upregulate the expression of surface proteins and secrete cytokines, both required for the optimal activation of CD4
and CD8
T cells. In this study, DC were cultured from 2-14 days in a rotary cell culture system, which generates a simulated microgravity (SMG) environment, and then the cells were assessed for maturation status and the capacity to activate T cells. Short-term culture (<72 h) of DC in SMG resulted in an increased expression of surface proteins associated with maturation and interleukin-6 production. Subsequently, the SMG exposed DC were superior to Static control DC at activating both CD4
and CD8
T cells as measured by interleukin-2 and interferon-γ production, respectively. However, long-term culture (4-14 d) of DC in SMG reduced the expression of maturation markers and the capacity to activate T cells as compared to Static DC controls.
The global and economic success of immunoglobulin-based therapeutics in treating a wide range of diseases has heightened the need to further enhance their efficacy and lifetime while diminishing ...deleterious side effects. The three most ubiquitous challenges of therapeutic immunoglobulin delivery are their relatively short lifetimes in vivo, the immunologic consequences of soluble antibody-antigen complexes, and the emergence of anti-drug antibodies. We describe the rapid, cell-tolerated chemical engineering of the erythrocyte membrane in order to display any antibody, our model system being the display of anti-Tumor Necrosis Factor (anti-TNFα), on the surface of long-lived red blood cells (RBCs) while masking the antibody's Fc region. We developed four synthetic approaches to generate RBC-Staphylococcal protein A (RBC-SpA) complexes: amino group targeting through N-hydrosuccinidyl ester-functionalized homobifunctional poly(ethylene glycol) (NHS-PEG-NHS), direct thiol group targeting using heterobifunctional NHS-PEG-maleimide (NHS-PEG-MAL), converted thiol targeting using heterobifunctional NHS-PEG-MAL, and click chemistry using heterobifunctional NHS-PEG-azido (NHS-PEG-N3) and NHS-PEG-alkyne (NHS-PEG-alk). The RBC-PEG-SpA complexes were formed within minutes, followed by the attachment of over 105 antibodies per RBC to the accessible RBC-bound SpA via Fc-Protein A coupling. The RBC-PEG-SpA-antibody arrays were shown to be stable for more than 60 days in PBS and for more than 42 days in serum containing buffer. RBC-PEG-SpA-antibody complexes were shown to remove TNFα from physiological buffer and had similar mechanical properties to unmodified RBCs. Out of the four approaches, the converted thiol method provided the most controlled chemistry and construct stability. We are now ideally positioned to determine the long-term in vivo efficacy of chemically membrane-engineered RBCs to remove antigens, like TNFα, from serum.
The global and economic success of immunoglobulin-based therapeutics in treating a wide range of diseases has heightened the need to further enhance their efficacy and lifetime while diminishing deleterious side effects. The three most ubiquitous challenges of therapeutic immunoglobulin delivery are their relatively short lifetimes in vivo, the immunologic consequences of soluble antibody-antigen complexes, and the emergence of anti-drug antibodies. We describe the rapid, cell-tolerated chemical engineering of the erythrocyte membrane to display any antibody, our model system being the display of anti-Tumor Necrosis Factor (anti-TNFα), on the surface of long-lived red blood cells (RBCs) while masking the antibody's Fc region. Conversion of RBCs into therapeutic delivery vehicles, we argue, would enhance the circulation life of immunoglobulin-based therapeutics while simultaneously evading deleterious immune response.
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Three new morpholine‐based ligands were synthesized and used in the ATRP reactions of free polymers and polymers grown from the surface of a protein in aqueous media. The new ligands were designed in ...a way that decreased their Lewis basicity with each additional incorporation of a morpholine group (1, 2, and 3). The morpholine‐based ligands significantly slowed the polymerization rates and led to lower dispersities than Et6TREN. Increasing the control of aqueous ATRP correlated well with decreasing ligand basicity.
Organophosphate nerve agents rapidly inhibit cholinesterases thereby destroying the ability to sustain life. Strong nucleophiles, such as oximes, have been used as therapeutic reactivators of ...cholinesterase‐organophosphate complexes, but suffer from short half‐lives and limited efficacy across the broad spectrum of organophosphate nerve agents. Cholinesterases have been used as long‐lived therapeutic bioscavengers for unreacted organophosphates with limited success because they react with organophosphate nerve agents with one‐to‐one stoichiometries. The chemical power of nucleophilic reactivators is coupled to long‐lived bioscavengers by designing and synthesizing cholinesterase‐polymer‐oxime conjugates using atom transfer radical polymerization and azide‐alkyne “click” chemistry. Detailed kinetic studies show that butyrylcholinesterase‐polymer‐oxime activity is dependent on the electrostatic properties of the polymers and the amount of oxime within the conjugate. The covalent coupling of oxime‐containing polymers to the surface of butyrylcholinesterase slows the rate of inactivation of paraoxon, a model nerve agent. Furthermore, when the enzyme is covalently inhibited by paraoxon, the covalently attached oxime induced inter‐ and intramolecular reactivation. Intramolecular reactivation will open the door to the generation of a new class of nerve agent scavengers that couple the speed and selectivity of biology to the ruggedness and simplicity of synthetic chemicals.
Butyrylcholinesterase (BChE)‐polymer‐oxime conjugates are synthesized by in situ atom transfer radical polymerization and azide‐alkyne “click” chemistry. The conjugate's activity depends on the electrostatic properties of the polymers and the oxime to BChE ratio. The oximes on the surface of BChE delay irreversible inhibition by paraoxon, and furthermore, are able to reactivate inhibited BChE through intra‐ and intermolecular interactions.
Cocaine perturbs mitovesicle biology in the brain D'Acunzo, Pasquale; Ungania, Jonathan M.; Kim, Yohan ...
Journal of extracellular vesicles,
January 2023, 2023-01-00, 20230101, 2023-01-01, Letnik:
12, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Cocaine, an addictive psychostimulant, has a broad mechanism of action, including the induction of a wide range of alterations in brain metabolism and mitochondrial homeostasis. Our group recently ...identified a subpopulation of non‐microvesicular, non‐exosomal extracellular vesicles of mitochondrial origin (mitovesicles) and developed a method to isolate mitovesicles from brain parenchyma. We hypothesised that the generation and secretion of mitovesicles is affected by mitochondrial abnormalities induced by chronic cocaine exposure. Mitovesicles from the brain extracellular space of cocaine‐administered mice were enlarged and more numerous when compared to controls, supporting a model in which mitovesicle biogenesis is enhanced in the presence of mitochondrial alterations. This interrelationship was confirmed in vitro. Moreover, cocaine affected mitovesicle protein composition, causing a functional alteration in mitovesicle ATP production capacity. These data suggest that mitovesicles are previously unidentified players in the biology of cocaine addiction and that target therapies to fine‐tune brain mitovesicle functionality may be beneficial to mitigate the effects of chronic cocaine exposure.
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