Ceria Nanoparticle Systems Alleviate Degenerative Changes in Mouse Postovulatory Aging Oocytes by Reducing Oxidative Stress and Improving Mitochondrial Functions.

Postovulatory aging oocytes usually feature diminished potential for fertilization and poor embryonic development due to enhanced oxidative damage to the subcellular organelles and macromolecules, which stands as a formidable obstacle in assisted reproductive technologies (ART). Here, we developed lipoic acid (LA) and polyethylene glycol (PEG)-modified CeO2 nanoparticles (LA-PEG-CeNPs) with biocompatibility, enzyme-like autocatalytic activity, and free radical scavenging capacity. We further investigated the LA-PEG-CeNPs effect in mouse postovulatory oocytes during in vitro aging. The results showed that LA-PEG-CeNPs dramatically reduced the accumulation of ROS in aging oocytes, improving mitochondrial dysfunction; they also down-regulated the pro-apoptotic activity by rectifying cellular caspase-3, cleaved caspase-3, and Bcl-2 levels. Consistently, this nanoenzyme prominently alleviated the proportion of abnormalities in spindle structure, chromosome alignment, microtubule stability, and filamentous actin (F-actin) distribution in aging oocytes, furthermore decreased oocyte fragmentation, and improved its ability of fertilization and development to blastocyst. Taken together, our finding suggests that LA-PEG-CeNPs can alleviate oxidative stress damage on oocyte quality during postovulatory aging, implying their potential value for clinical practice in assisted reproduction.

Ångstrom-scale gold particles loaded with alendronate via alpha-lipoic acid alleviate bone loss in osteoporotic mice.

Osteoporosis is a highly prevalent metabolic disease characterized by low systemic bone mass and deterioration of bone microarchitecture, resulting in reduced bone strength and increased fracture risk. Current treatment options for osteoporosis are limited by factors such as efficacy, cost, availability, side effects, and acceptability to patients. Gold nanoparticles show promise as an emerging osteoporosis therapy due to their osteogenic effects and ability to allow therapeutic delivery but have inherent constraints, such as low specificity and the potential for heavy metal accumulation in the body. This study reports the synthesis of ultrasmall gold particles almost reaching the Ångstrom (Ång) dimension. The antioxidant alpha-lipoic acid (LA) is used as a dispersant and stabilizer to coat Ångstrom-scale gold particles (AuÅPs). Alendronate (AL), an amino-bisphosphonate commonly used in drug therapy for osteoporosis, is conjugated through LA to the surface of AuÅPs, allowing targeted delivery to bone and enhancing antiresorptive therapeutic effects. In this study, alendronate-loaded Ångstrom-scale gold particles (AuÅPs-AL) were used for the first time to promote osteogenesis and alleviate bone loss through regulation of the WNT signaling pathway, as shown through in vitro tests. The in vivo therapeutic effects of AuÅPs-AL were demonstrated in an established osteoporosis mouse model. The results of Micro-computed Tomography, histology, and tartrate-resistant acid phosphatase staining indicated that AuÅPs-AL significantly improved bone density and prevented bone loss, with no evidence of nanoparticle-associated toxicity. These findings suggest the possible future application of AuÅPs-AL in osteoporosis therapy and point to the potential of developing new approaches for treating metabolic bone diseases using Ångstrom-scale gold particles.

Discovery of neuroprotective Agents: Potent, brain Penetrating, lipoic acid derivatives for the potential treatment of ischemic stroke by regulating oxidative stress and inflammation - a Preliminary study.

Stroke poses a serious risk to the physical and mental health of patients. Endogenous compounds are widely used to treat ischemic stroke. Lipoic acid, a naturally occurring (R)-5-(1,2-dithiolan-3-yl)pentanoic acid, has therapeutic potential for the treatment of ischemic stroke. However, the direct application of lipoic acid is limited by its relatively low efficacy and instability. Therefore, there is a need to modify the structure of lipoic acid to improve its pharmaceutical capabilities. Currently, 37 lipoic acid derivatives have been synthesized, and compound AA-9 demonstrated optimal therapeutic potential in an in vitro model of induced oxidative damage using tert-butyl hydroperoxide (t-BHP). In addition, in vitro experiments have shown that compound AA-9 has an excellent safety profile. Subsequently, the therapeutic effect of AA-9 was significant in the rat MCAO ischemic stroke model, which may be attributed to the antioxidant and anti-inflammatory effects of compound AA-9 by activating PGC-1α and inhibiting NLRP3. Notably, compound AA-9 exhibited higher stability and better bioavailability properties than ALA in plasma stability and pharmacokinetic properties. In conclusion, AA-9 may be a promising neuroprotective agent for the treatment of ischemic stroke and warrants further investigation.

Low-Triggering-Potential and Narrow-Potential-Window Electrochemiluminescence of Silver Nanoclusters for Gene Assay.

A low-triggering potential and a narrow-potential window are anticipated to decrease the electrochemical interference and cross talk of electrochemiluminescence (ECL). Herein, by exploiting the low oxidative potential (0.82 V vs Ag/AgCl) of dihydrolipoic acid-capped sliver nanoclusters (DHLA-AgNCs), a coreactant ECL system of DHLA-AgNCs/hydrazine (N2H4) is proposed to achieve efficient and oxidative-reduction ECL with a low-triggering potential of 0.82 V (vs Ag/AgCl) and a narrow-potential window of 0.22 V. The low-triggering-potential and narrow-potential-window nature of ECL can be primarily preserved upon labeling DHLA-AgNCs to probe DNA and immobilizing DHLA-AgNCs onto the Au surface via sandwiched hybridization, which eventually enables a selective ECL strategy for the gene assay at +0.82 V. This gene assay strategy can sensitively determine the gene of human papillomavirus from 10 to 1000 pM with a low limit of detection of 5 pM (S/N = 3) and would open a way to improve the applied ECL bioassay.

Biological applications of lipoic acid-based polymers: an old material with new promise.

Lipoic acid (LA) is a versatile antioxidant that has been used in the treatment of various oxidation-reduction diseases over the past 70 years. Owing to its large five-membered ring tension, the dynamic disulfide bond of LA is highly active, enabling the formation of poly(lipoic acid) (PLA) via ring-opening polymerization (ROP). Herein, we first summarize disulfide-mediated ROP polymerization strategies, providing basic routes for designing and preparing PLA-based materials. PLA, as a biologically derived, low toxic, and easily modified material, possesses dynamic disulfide bonds and universal non-covalent carboxyl groups. We also shed light on the biomedical applications of PLA-based materials based on their biological and structural features and further divide recent works into six categories: antibacterial, anti-inflammation, anticancer, adhesive, flexible electronics, and 3D-printed tissue scaffolds. Finally, the challenges and future prospects associated with the biomedical applications of PLA are discussed.

Solubilization and stabilization of lipoic acid trisulfide by creation of various ß-cyclodextrin clathrates.

Lipoic acid trisulfide, a sulfane sulfur-containing trisulfide of α-lipoic acid, holds promise in pharmaceuticals, yet knowledge gaps persist regarding its synthesis, properties, and stability. Here, we synthesized the lipoic acid trisulfide with a purity exceeding 99% from α-lipoic acid on a gram scale and obtained novel ß-cyclodextrin clathrates (84%-95% yield). Differential scanning calorimetry confirmed the inclusion of lipoic acid trisulfide in ß-cyclodextrins. The resulting ß-cyclodextrin clathrates exhibited significant improvements in water solubility and thermal stability. This pioneering study demonstrated a novel approach to the practical preparation of trisulfide and its ß-cyclodextrin clathrates as active ingredients, paving the way for clinical development.

Lipoic acid-mediated oral drug delivery system utilizing changes on cell surface thiol expression for the treatment of diabetes and inflammatory diseases.

Lipoic acid (LA), which has good safety and oral absorption, is obtained from various plant-based food sources and needs to be supplemented through human diet. Moreover, substances with a disulfide structure can enter cells through dynamic covalent disulfide exchange with thiol groups on the cell membrane surface. Based on these factors, we constructed LA-modified nanoparticles (LA NPs). Our results showed that LA NPs can be internalized into intestinal epithelial cells through surface thiols, followed by intracellular transcytosis via the endoplasmic reticulum-Golgi pathway. Further mechanistic studies indicated that disulfide bonds within the structure of LA play a critical role in this transport process. In a type I diabetes rat model, the oral administration of insulin-loaded LA NPs exhibited a more potent hypoglycemic effect, with a pharmacokinetic bioavailability of 5.42 ± 0.53%, representing a 1.6 fold enhancement compared to unmodified PEG NPs. Furthermore, a significant upregulation of surface thiols in inflammatory macrophages was reported. Thus, we turned our direction to investigate the uptake behavior of inflammatory macrophages with increased surface thiols towards LA NPs. Inflammatory macrophages showed a 2.6 fold increased uptake of LA NPs compared to non-inflammatory macrophages. Surprisingly, we also discovered that the antioxidant resveratrol facilitates the uptake of LA NPs in a concentration-dependent manner. This is mainly attributed to an increase in glutathione, which is involved in thiol uptake. Consequently, we employed LA NPs loaded with resveratrol for the treatment of colitis and observed a significant alleviation of colitis symptoms. These results suggest that leveraging the variations of thiol expression levels on cell surfaces under both healthy and diseased states through an oral drug delivery system mediated by the small-molecule nutrient LA can be employed for the treatment of diabetes and certain inflammatory diseases.

Biodegradable Lipid-Modified Poly(Guanidine Thioctic Acid)s: A Fortifier of Lipid Nanoparticles to Promote the Efficacy and Safety of mRNA Cancer Vaccines.

Lipid nanoparticles (LNPs)-based messenger RNA (mRNA) therapeutics have emerged with promising potentials in the fields of infectious diseases, cancer vaccines, and protein replacement therapies; however, their therapeutic efficacy and safety can still be promoted by the optimization of LNPs formulations. Unfortunately, current LNPs suffer from increased production of reactive oxygen species during translation, which leads to a decreased translation efficiency and the onset of inflammation and other side effects. Herein, we synthesize a lipid-modified poly(guanidine thioctic acid) polymer to fabricate novel LNPs for mRNA vaccines. The acquired G-LNPs significantly promote the translation efficiency of loaded mRNA and attenuate inflammation after vaccination through the elimination of reactive oxygen species that are responsible for translational inhibition and inflammatory responses. In vivo studies demonstrate the excellent antitumor efficacy of the G-LNPs@mRNA vaccine, and two-dose vaccination dramatically increases the population and infiltration of cytotoxic T cells due to the intense antitumor immune responses, thus generating superior antitumor outcomes compared with the mRNA vaccine prepared from traditional LNPs. By synergy with immune checkpoint blockade, the tumor inhibition of G-LNPs@mRNA is further boosted, indicating that G-LNPs-based mRNA vaccines will be powerful and versatile platforms to combat cancer.

Dynamic Antimicrobial Poly(disulfide) Coatings Exfoliate Biofilms On Demand Via Triggered Depolymerization.

Bacterial biofilms are notoriously problematic in applications ranging from biomedical implants to ship hulls. Cationic, amphiphilic antibacterial surface coatings delay the onset of biofilm formation by killing microbes on contact, but they lose effectiveness over time due to non-specific binding of biomass and biofilm formation. Harsh treatment methods are required to forcibly expel the biomass and regenerate a clean surface. Here, a simple, dynamically reversible method of polymer surface coating that enables both chemical killing on contact, and on-demand mechanical delamination of surface-bound biofilms, by triggered depolymerization of the underlying antimicrobial coating layer, is developed. Antimicrobial polymer derivatives based on α-lipoic acid (LA) undergo dynamic and reversible polymerization into polydisulfides functionalized with biocidal quaternary ammonium salt groups. These coatings kill >99.9% of Staphylococcus aureus cells, repeatedly for 15 cycles without loss of activity, for moderate microbial challenges (≈105 colony-forming units (CFU) mL-1, 1 h), but they ultimately foul under intense challenges (≈107 CFU mL-1, 5 days). The attached biofilms are then exfoliated from the polymer surface by UV-triggered degradation in an aqueous solution at neutral pH. This work provides a simple strategy for antimicrobial coatings that can kill bacteria on contact for extended timescales, followed by triggered biofilm removal under mild conditions.

Synthesis and glutathione peroxidase (GPx)-like activity of selenocystine (SeC) bioconjugates of biotin and lipoic acid.

The conjugation of active biomolecules provides insight into their bioreactivity, leading to many applications in biotechnology and materials science. Herein, we report L-selenocystine (SeC) bioconjugates of lipoic acid (universal antioxidant) and biotin (Vitamin-H). The SeC-bioconjugates, SeC-Biotin (1) and SeC-Lipoic acid (2) were synthesized using solid phase peptide synthesis (SPPS) method and were characterized by multinuclear 1D (1H, 13C, 77Se) and 2D (1H-1H COSY and 1H-13C TOCSY) NMR spectroscopy, ESI-MS spectrometry, and RP-HPLC. The GPx-like enzyme mimicking activity of the SeC-bioconjugates 1 and 2 has been investigated through the coupled reductase assay method for the catalytic reductions of hydrogen peroxide into water. A significant enhancement in GPx-like enzymatic activity was observed for both novel bioconjugates SeC-Biotin (1) and SeC-Lipoic acid (2) as compared to diphenyl diselenide (Ph2Se2), L-selenocystine (SeC), biotin, lipoic acid, and ebselen.

Redox-sensitive self-assembled micelles based on low molecular weight chitosan-lipoic acid conjugates for the delivery of doxorubicin: Effect of substitution degree of lipoic acid.

Amphiphilic low molecular weight chitosan-lipoic acid (LC-LA) conjugates with different degrees of substitution (DS) of LA were synthesized by N, N'­carbonyldiimidazole (CDI) catalysis to self-assemble into redox-sensitive micelles. Critical micelle concentration (CMC), size, zeta potential, biocompatibility and redox-sensitive behavior of blank micelles were investigated. The results indicated that blank micelles with low CMC, nanoscale size and positive zeta potential showed excellent biocompatibility and redox-sensitive behavior. Doxorubicin (Dox) loaded micelles were prepared by encapsulating Dox into blank micelles. The loading ability, trigger-release behavior, antitumor activity and cellular uptake of Dox loaded micelles were studied. The results demonstrated that Dox loaded micelles with superior loading ability exhibited redox-trigger behavior, strong antitumor activity and increased cellular uptake efficiency against A549 cell. Besides, the effect of DS of LA on above properties was estimated. An increase in DS of LA reduced the CMC and cumulative release amount of Dox, but improved the loading efficiency, antitumor activity, and cellular uptake of Dox loaded micelles, which resulted from stronger interaction of hydrophobic groups in micelles with the DS of LA increased. Overall, self-assembled LC-LA micelles with good biosecurity and redox-sensitive behavior hold promising application prospects in Dox delivery and improving cancer therapeutic effect of Dox.

Bioderived Lipoic Acid-Based Dynamic Covalent Nanonetworks of Poly(disulfide)s: Enhanced Encapsulation Stability and Cancer Cell-Selective Delivery of Drugs.

Dynamic covalent poly(disulfide)-based cross-linked nanoaggregates, termed nanonetworks (NNs), endowed with pH- and redox-responsive degradation features have been fabricated for stable noncovalent encapsulation and triggered cargo release in a controlled fashion. A bioderived lipoic acid-based Gemini surfactant-like amphiphilic molecule was synthesized for the preparation of nanoaggregates. It self-assembles by a entropy-driven self-assembly process in aqueous milieu. To further stabilize the self-assembled nanostructure, the core was cross-linked by ring-opening disulfide exchange polymerization (RODEP) of 1,2-dithiolane rings situated inside the core of the nanoaggregates. The cross-linked nanoaggregates, i.e., nanonetwork, are found to be stable in the presence of blood serum, and also, they maintain the self-assembled structure even below the critical aggregation concentration (CAC) as probed by dynamic light scattering (DLS) experiments. The nanonetwork showed almost 50% reduction in guest leakage compared to that of the nanoaggregates as shown by the release profile in the absence of stimuli, suggesting high encapsulation stability as evidenced by the fluorescence resonance energy transfer (FRET) experiment. The decross-linking of the nanonetwork occurs in response to redox and pH stimuli due to disulfide reduction and ß-thioester hydrolysis, respectively, thus empowering disassembly-mediated controlled cargo release up to ∼87% for 55 h of incubation. The biological evaluation of the doxorubicin (DOX)-loaded nanonetwork revealed environment-specific surface charge modulation-mediated cancer cell-selective cellular uptake and cytotoxicity. The benign nature of the nanonetwork toward normal cells makes the system very promising in targeted drug delivery applications. Thus, the ease of synthesis, nanonetwork fabrication reproducibility, robust stability, triggered drug release in a controlled fashion, and cell-selective cytotoxicity behavior, we believe, will make the system a potential candidate in the development of robust materials for chemotherapeutic applications.

Understanding α-lipoic acid photochemistry helps to control the synthesis of plasmonic gold nanostructures.

We propose the photopolymerization of lipoic acid (LA) as an novel approach to produce a cross-linked polymeric matrix of lipoic acid monomers (PALA) which helps to control the size of plasmonic gold nanostructures when using 3,3,6,8-tetramethyl-1-tetralone as the photo-initiator for the reduction of Au(III) to Au0. A complete characterization of the polymer is included, and the dual behaviour of LA as an in situ stabilizer and reducing agent is investigated. These findings are relevant to the understanding of the photochemical transformation of this biologically relevant compound and would benefit the increasing use of LA and PALA for the synthesis of various nanomaterials.

Review of lipoic acid: From a clinical therapeutic agent to various emerging biomaterials.

Lipoic acid (LA), an endogenous small molecule in organisms, has been extensively used for the highly efficient clinical treatment of malignant diseases, which include diabetes, Alzheimer's disease, and cancer over the past seven decades. Tremendous progresses have been made on the use of LA in nanomedicine for the development of various biomaterials because of its unique biological properties and highly adaptable structure since the first discovery. However, there are few reviews thus far, to our knowledge, summarizing this hot subject of research of LA and its derived biomaterials. For this purpose, we present herein the first comprehensive summary on the design and development of LA and its derived materials for biomedical applications. This review first discusses the therapeutic use of LA followed by the description of synthesis and preclinical study of LA-derived-small molecules. The applications of various LA and poly (lipoic acid) (PLA)-derived-biomaterials are next summarized in detail with an emphasis on the use of LA for the design of biomaterials and the diverse properties. This review describes the development of LA from a clinical therapeutic agent to a building unit of various biomaterials field, which will promote the further discovery of new therapeutic uses of LA as therapeutic agents and facile development of LA-based derivates with greater performance for biomedical applications.

Strategic application of liposomal system to R-α-lipoic acid for the improvement of nutraceutical properties.

R-α-lipoic acid (RLA) and dihydrolipoic acid (DHLA), a reduced form of RLA, are potent endogenous antioxidants that can reduce oxidative damage. Despite their numerous nutraceutical potentials, clinical applications of RLA are still limited due to its poor solubility and stability problems. This study aimed to develop an RLA-loaded liposome (LIP/RLA) for the improvement of nutraceutical properties. LIP/RLA was developed by a typical solvent injection method. Uniform liposomes of LIP/RLA were observed by transmission electron microscopy, and the mean particle size was calculated to be ∼150 nm from the data of dynamic light scattering. LIP/RLA could prevent the degradation of RLA even under acidic conditions (pH 1.2) possibly due to the encapsulation of RLA into the liposomal structure. In the release test under pH6.8 with lipase, LIP/RLA showed relatively rapid release of RLA, possibly due to the lipolysis of phospholipids by lipase. After the oral administration of LIP/RLA (10 mg-RLA/kg, p.o.) in rats, the systemic exposures of RLA and DHLA increased by 2.8- and 5.8-fold, respectively. In a rat model of acute hepatic injury induced by carbon tetrachloride (CCl4) (0.7 mL-CCl4/kg, p.o.), orally dosed LIP/RLA (3 mg-RLA/kg, p.o.) resulted in 78.7% and 86.4% reductions of plasma alanine aminotransferase, and aspartate aminotransferase, respectively; however, RLA was found to be less effective possibly due to the poor oral absorption. The RLA-loaded liposomal system might be a promising carrier for poorly water-soluble materials with poor stability under acidic conditions, as well as RLA, to improve their oral absorption and nutraceutical properties.

Further assessments of ligase LplA-mediated modifications of proteins in vitro and in cellulo.

BACKGROUND: Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules. METHODS AND RESULTS: In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N3-lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach. CONCLUSIONS: In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach-the Halo Tag-to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology.

A molybdenum oxide-based degradable nanosheet for combined chemo-photothermal therapy to improve tumor immunosuppression and suppress distant tumors and lung metastases.

Molybdenum oxide (MoOx) nanosheets have drawn increasing attention for minimally invasive cancer treatments but still face great challenges, including complex modifications and the lack of efficient accumulation in tumor. In this work, a novel multifunctional degradable FA-BSA-PEG/MoOx nanosheet was fabricated (LA-PEG and FA-BSA dual modified MoOx): the synergistic effect of PEG and BSA endows the nanosheet with excellent stability and compatibility; the FA, a targeting ligand, facilitates the accumulation of nanosheets in the tumor. In addition, DTX, a model drug for breast cancer treatment, was loaded (76.49%, 1.5 times the carrier weight) in the nanosheets for in vitro and in vivo antitumor evaluation. The results revealed that the FA-BSA-PEG/MoOx@DTX nanosheets combined photothermal and chemotherapy could not only inhibit the primary tumor growth but also suppress the distant tumor growth (inhibition rate: 51.7%) and lung metastasis (inhibition rate: 93.6%), which is far more effective compared to the commercial Taxotere®. Exploration of the molecular mechanism showed that in vivo immune response induced an increase in positive immune responders, suppressed negative immune suppressors, and established an inflammatory tumor immune environment, which co-contributes towards effective suppression of tumor and lung metastasis. Our experiments demonstrated that this novel multifunctional nanosheet is a promising platform for combined chemo-photothermal therapy.

Quaternary Ammonium Salts Anchored on Cross-Linked (R)-(+)-Lipoic Acid Nanoparticles for Drug-Resistant Tumor Therapy.

A membrane-lytic mechanism-based nanodrug is developed for drug-resistant tumor therapy by anchoring the small-molecule quaternary ammonium salt (QAS) on cross-linked (R)-(+)-lipoic acid nanoparticles (cLANs). The anchoring of QAS on the nanoparticle avoids the direct attack of long alkyl chains to the cell membrane under physiological conditions, while after entering tumor cells, the QAS is released from the dissociated cLANs, migrates to the phospholipid bilayer via electrostatic interaction, and destroys the cell membrane by the puncture of long alkyl chains. Since the QAS is designed to finally be hydrolyzed to amino acid betaine and food additive cetanol and the cLANs degrade to dihydrolipoic acid (DHLA, reduced form of dietary antioxidant lipoic acid in cells), the QAS@cLANs hold superior biosafety. In addition to the drug-resistant tumors, the QAS@cLANs demonstrate significant inhibition of metastatic tumors. This work provides not only a general and clinic-promising treatment for the refractory tumors but also opens a door for the medicinal use of QAS.

Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion.

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.

Lipoate-acid ligase a modification of native antibody: Synthesis and conjugation site analysis.

Bioconjugation is an important chemical biology research focus, especially in the development of methods to produce pharmaceutical bioconjugates and antibody-drug conjugates (ADCs). In this report, an enzyme-catalyzed conjugation method combined with a chemical reaction was used to modify a native antibody under mild reaction conditions. Our investigation revealed that lipoic-acid ligase (LplA) modifies native IgG1 with biased site-specificity. An intact mass analysis revealed that 98.3% of IgG1 was modified by LplA and possessed at least one molecule of octanocic acid. The average number of modifications per antibody was calculated to be 4.6. Peptide mapping analysis revealed that the modified residues were K225, K249 and K363 in the Fc region, and K30, K76 and K136 in the heavy chain and K39/K42, K169, K188 and K190 in the light chain of the Fab region. Careful evaluation including solvent exposed amino acid analysis suggested that these conjugate sites were not only solvent exposed but also biased by the site-specificity of LplA. Furthermore, antibody fragment conjugation may be able to take advantage of this enzymatic approach. This feasibility study serves as a demonstration for preparing enzymatically modified antibodies with conjugation site analysis.