ABSTRACT
The trans-decalin structure formed by intramolecular Diels-Alder cycloaddition is widely present among bioactive natural products isolated from fungi. We elucidated the concise three-enzyme biosynthetic pathway of the cytotoxic myceliothermophin and biochemically characterized the Diels-Alderase that catalyzes the formation of trans-decalin from an acyclic substrate. Computational studies of the reaction mechanism rationalize both the substrate and stereoselectivity of the enzyme.
Subject(s)
Eukaryota/chemistry , Naphthalenes/metabolism , Peptide Synthases/metabolism , Polyketide Synthases/metabolism , Biocatalysis , Cycloaddition Reaction , Eukaryota/metabolism , Euryarchaeota/enzymology , Naphthalenes/chemistry , Peptide Synthases/chemistry , Polyketide Synthases/chemistryABSTRACT
Dimeric indole alkaloids are structurally diverse natural products that have attracted significant attention from the synthetic and biosynthetic communities. Here, we describe the characterization of a P450 monooxygenase CnsC from Penicillium that catalyzes the heterodimeric coupling between two different indole moieties, tryptamine and aurantioclavine, to construct vicinal quaternary stereocenters and yield the heptacyclic communesin scaffold. We show, via biochemical characterization, substrate analogues, and computational methods that CnsC catalyzes the C3-C3' carbon-carbon bond formation and controls the regioselectivities of the pair of subsequent aminal bond formations to yield the communesin core. Use of ω-N-methyltryptamine and tryptophol in place of tryptamine led to the enzymatic synthesis of isocommunesin compounds, which have not been isolated to date.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/metabolism , Indoles/metabolism , Cytochrome P-450 Enzyme System/chemistry , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Indoles/chemistry , Isomerism , Molecular StructureABSTRACT
Encapsulating anticancer protein therapeutics in nanocarriers is an attractive option to minimize active drug destruction, increase local accumulation at the disease site, and decrease side effects to other tissues. Tumor-specific ligands can further facilitate targeting the nanocarriers to tumor cells and reduce nonspecific cellular internalization. Rationally designed non-covalent protein nanocapsules incorporating copper-free "click chemistry" moieties, polyethylene glycol (PEG) units, redox-sensitive cross-linker, and tumor-specific targeting ligands were synthesized to selectively deliver intracellular protein therapeutics into tumor cells via receptor-mediated endocytosis. These nanocapsules can be conjugated to different targeting ligands of choice, such as anti-Her2 antibody single-chain variable fragment (scFv) and luteinizing hormone releasing hormone (LHRH) peptide, resulting in specific and efficient accumulation within tumor cells overexpressing corresponding receptors. LHRH-conjugated nanocapsules selectively delivered recombinant human tumor suppressor protein p53 and its tumor-selective supervariant into targeted tumor cells, which led to reactivation of p53-mediated apoptosis. Our results validate a general approach for targeted protein delivery into tumor cells using cellular-responsive nanocarriers, opening up new opportunities for the development of intracellular protein-based anticancer treatment.
Subject(s)
Drug Carriers/chemistry , Nanocapsules/chemistry , Recombinant Proteins/chemistry , Tumor Suppressor Protein p53/chemistry , Amino Acid Sequence , Azides/chemistry , Cell Survival/drug effects , Click Chemistry , Drug Carriers/metabolism , Drug Carriers/toxicity , Drug Liberation , Gonadotropin-Releasing Hormone/chemistry , HeLa Cells , Humans , Ligands , Nanocapsules/toxicity , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Polyethylene Glycols/chemistry , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/metabolism , Surface PropertiesABSTRACT
Proteins play a crucial role in life, taking part in all vital processes in the body. In the past decade, there was increasing interest in delivering active forms of proteins to specific cells and organs. Intracellular protein delivery holds enormous promise for biological and medical applications, including cancer therapy, vaccination, regenerative medicine, treatment for loss-of-function genetic diseases and imaging. This tutorial review surveys recent developments in intracellular protein delivery using various nanocarriers. Methods such as lipid-mediated colloidal systems, polymeric nanocarriers, inorganic systems and protein-mediated carriers are reviewed. Advantages and limitations of current strategies, as well as future opportunities and challenges are also discussed.
Subject(s)
Drug Carriers/chemistry , Intracellular Space/metabolism , Nanostructures/chemistry , Proteins/metabolism , Animals , Cell Line , Humans , Protein Transport , Proteins/chemistryABSTRACT
Direct delivery of proteins to the cytosol of cells holds tremendous potential in biological and medical applications. Engineering vehicles for escorting proteins to the cytosol in a controlled release fashion has thus generated considerable interest. We report here the preparation of redox-responsive single-protein nanocapsules for intracellular protein delivery. Through in situ interfacial polymerization, the target protein is noncovalently encapsulated into a positively-charged polymeric shell interconnected by disulfide-containing crosslinkers. The dissociation of the polymeric shell under reducing conditions and the subsequent release of protein were confirmed using cell-free assays in the presence of glutathione (GSH). The nanocapsules were demonstrated to be efficiently internalized into the cells and to release the protein in the reducing cytosol. Using the nanocapsule as a vehicle, we showed that active caspase 3 (CP-3) can be delivered and can induce apoptosis in a variety of human cancer cell lines, including HeLa, MCF-7 and U-87 MG. Our approach therefore presents an effective intracellular protein delivery strategy for therapeutic, diagnostic and reprogramming applications.
Subject(s)
Drug Carriers/chemistry , Drug Delivery Systems , Nanocapsules/chemistry , Oxidation-Reduction , Proteins/chemistry , Proteins/metabolism , Apoptosis , Cell Line , Endocytosis/physiology , Humans , Materials Testing , Molecular Structure , Nanocapsules/adverse effectsABSTRACT
Mercury is a highly hazardous and widespread pollutant with bioaccumulative properties. Novel approaches that meet the criteria of desired selectivity, high sensitivity, good biocompatibility, and low background interference in natural settings are continuously being explored. We herein describe a new strategy utilizing the combination of infrared fluorescent protein (IFP) and its chromophore as an infrared fluorescence probe for mercury ion (Hg(II)) detection. Hg(II) has been validated to have specific binding affinity to a cysteine residue (C24) of IFP, thereby inhibiting the conjugation of IFP chromophore biliverdin (BV) to C24 and "turning off" the infrared emission of IFP. The IFP/BV sensor has high selectivity toward Hg(II) among other metal ions over a broad pH range. The in vitro detection limit was determined to be less than 50 nM. As a genetically encoded probe, we demonstrate the IFP/BV sensor can serve as a tool to detect Hg(II) in living organisms or tissues. Moreover, we have exploited a protein-agarose hydrogel-based paper assay to immobilize IFP for detection of Hg(II) in a portable and robust fashion.
Subject(s)
Biosensing Techniques/instrumentation , Hydrogels/chemistry , Hydrogels/metabolism , Luminescent Proteins/chemistry , Luminescent Proteins/metabolism , Mercury/analysis , Paper , Biliverdine/metabolism , Binding, Competitive , Cysteine/metabolism , HEK293 Cells , Humans , Mercury/metabolism , Models, Molecular , Protein Conformation , Reproducibility of Results , Spectrophotometry, InfraredABSTRACT
Proteins possess distinct intracellular roles allowing them to have vast therapeutic applications. However, due to poor cellular permeability and fragility of most proteins, intracellular delivery of native, active proteins is challenging. We describe a biomimetic protein delivery vehicle which is degradable upon the digestion by furin, a ubiquitous intracellular protease, to release encapsulated cargos. Proteins were encapsulated in a nanosized matrix prepared with monomers and a bisacrylated peptide cross-linker which can be specifically recognized and cleaved by furin. Release of encapsulated protein was confirmed in a cell-free system upon proteolytic degradation of nanocapsules. In vitro cell culture studies demonstrated successful intracellular delivery of both nuclear and cytosolic proteins and confirmed the importance of furin-degradable construction for native protein release. This endoprotease-mediated intracellular delivery system may be extended to effectively deliver various biological therapeutics.
Subject(s)
Biomimetic Materials , Furin/metabolism , Intracellular Space/metabolism , Nanocapsules , Proteins/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , HeLa Cells , Humans , Mice , Models, Molecular , Protein Conformation , Protein Transport , Proteins/chemistryABSTRACT
Atomic compositions and molar extinction coefficients of PbSe semiconductor nanocrystals were determined by atomic absorption spectrometry, UV-vis-NIR spectrophotometry, and transmission electron microscopy. The Pb/Se atomic ratio was found to be size-dependent with a systematic excess of Pb atoms in the PbSe nanocrystal system. Experimental results indicated that the individual PbSe nanocrystal was nonstoichiometric, consisting of a PbSe core and an extra layer of Pb atoms. For these nonstoichiometric PbSe semiconductor nanocrystals, we proposed a new computational approach to calculate the total number of Pb and Se atoms in different sized particles. This calculation played a key role on the accurate determination of the strongly size-dependent extinction coefficient, which followed a power law with an exponent of approximately 2.5.
