Synthetic Linker Research Articles

Innovative epitopes in Staphylococcal Protein-A an immuno-informatics approach to combat MDR-MRSA infections

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge in clinical environments due to its resistance to standard antibiotics. Staphylococcal Protein A (SpA), a crucial virulence factor of MRSA, undermines host immune responses, making it an attractive target for vaccine development. This study aimed to identify potential epitopes within SpA that could elicit robust immune responses, ultimately contributing to the combat against multidrug-resistant (MDR) MRSA.MethodsThe SpA protein sequence was retrieved from the UniProt database, with various bioinformatics tools employed for epitope prediction. T-cell epitopes were identified using the Tepitool server, focusing on high-affinity interactions with prevalent human leukocyte antigens (HLAs). B-cell epitopes were predicted using the BepiPred tool. Predicted epitopes underwent docking studies with HLA molecules to evaluate binding properties. In-silico analyses confirmed the antigenicity, promiscuity, and non-glycosylated nature of the selected epitopes.ResultsSeveral T and B cell epitopes within SpA were identified, showcasing high binding affinities and extensive population coverage. A multi-epitope vaccine construct, linked by synthetic linkers and an adjuvant, was modelled, refined, and validated through various bioinformatics assessments. The vaccine candidate was subsequently docked with Toll-like receptor 4 (TLR-4) to evaluate its potential for immunogenicity.ConclusionThis study lays the groundwork for developing epitope-based vaccines targeting SpA in MRSA, identifying promising candidates for experimental validation and contributing to innovative immunotherapeutic strategies against MRSA infections.

Construction of homologous branched oligomer megamolecules based on linker-directed protein assembly.

Utilizing the building blocks of recombinant proteins and synthetic linkers, we have obtained two distinct octameric megamolecules with diverse branched structures. This approach combines principles from both click chemistry and protein engineering technology, enabling the integration of functional domains within highly ordered protein assemblies for biomedical applications.

Synthetic Amine Linkers for Efficient Sortagging.

Enzymatic site-specific bioconjugation techniques, in particular sortase-mediated ligation, are increasingly used to generate conjugated proteins for a wide array of applications. Extension of the utility and practicality of sortagging for diverse purposes is critically dependent on further improvement of the efficiency of sortagging reactions with a wider structural variety of substrates. We present a comprehensive comparative mass spectrometry screening study of synthetic nonpeptidic incoming amine nucleophile substrates of Staphylococcus aureus Sortase A enzyme. We have identified the optimal structural motifs among the chemically diverse set of 452 model primary and secondary amine-containing sortagging substrates, and we demonstrate the utility of representative amine linkers for efficient C-terminal biotinylation of nanobodies.

Abstract 5760: Intra-tissue quantitative analysis using LC/MS will determine the mechanisms of interstitial pneumonia induced by antibody-drug conjugates

Abstract Background: Antibody-drug conjugates (ADCs) are a new class of therapeutics that combine a monoclonal antibody with a cytotoxic agent (known as their payloads) via a synthetic linker. While ADCs are one of game changer and are the fastest growing classes with novel clinical benefits recently, interstitial pneumonia (ILD) is a clinical problem as a fatal adverse event. However, the mechanism of ADC-induced ILD is under investigation. Purpose: We clarify ADC-ILD mechanism by analyzing released payload distribution in the lung using a novel Trophoblast cell surface antigen 2 (TROP2)-targeting ADC. Material and Methods: TROP2-eribulin is a novel TROP2-targeting ADC composed of the TROP2 antibody and cytotoxic payload eribulin conjugated by a cleavable linker. Four human cancer cell lines (A549, HeLa, NCI-H2110 and SKOV3) and one human- leukemia monocytic cell line (THP-1) were used in this study. Cell surface TROP2 antigen expression was evaluated by flow cytometry. We investigated the concentration and distribution of the TROP2-eribulin and the payload released from the TROP2-eribulin in tumor and lung tissue, using TROP2 low and high expression human cancer xenograft model that were treated with various doses of TROP2-eribulin. The concentration of erbulin was analyzed by liquid chromatograph mass spectrometry, and tissue distribution of ADCs was assessed by immunofluorescence (IF) assay. Results: In vitro analysis showed that the concentration of intracellularly released eribulin was significantly higher in NCI-H2110 with high TROP2 expression compared to A549 with low TROP2 expression (P < 0.05). In vivo analysis, the concentration of released eribulin in tumor tissue was approximately 10-fold higher in the NIC-H2110 xenograft model than the A549 xenograft model, while analysis of lung tissue showed that TROP2-eribulin was distributed in lung tissue in a dose-dependent manner of TROP2-eribulin regardless of TROP2 expression, with significantly more eribulin released in the high-dose group of TROP2-eribulin than in the other dose groups (P < 0.05). IF assay showed that TROP2-eribuilin localized to alveolar macrophages. In the analysis using THP-1 cell, which mimic macrophage function, the concentration of eribulin released from TROP2-eribuilin was significantly reduced by the use of an Fc receptor inhibitor (P < 0.05). Conclusions: Nonspecific uptake of ADC by alveolar macrophages via Fcγ receptors releases cytotoxic payload into lung tissue, regardless of tumor antigen expression levels. This understanding can help elucidate the pathogenesis of ADC-induced ILD. Citation Format: Shigehiro Koganemaru, Hirobumi Fuchigami, Chihiro Morizono, Hiroko Shinohara, Yasutoshi Kuboki, Keiji Furuuchi, Toshimitsu Uenaka, Toshihiko Doi, Masahiro Yasunaga. Intra-tissue quantitative analysis using LC/MS will determine the mechanisms of interstitial pneumonia induced by antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5760.

Bending the Rules: Amplifying PD-L1 Immunoregulatory Function Through Flexible Polyethylene Glycol Synthetic Linkers.

Immune checkpoint signaling, such as programmed cell death protein-1 (PD-1), is a key target for immunotherapy due to its role in dampening immune responses. PD-1 signaling in T cells is regulated by complex physicochemical and mechanical cues. However, how these mechanical forces are integrated with biochemical responses remains poorly understood. Our previous work demonstrated that the use of an immobilizing polyethylene glycol (PEG) linker on synthetic microgels for the presentation of a chimeric form of PD-L1, SA-PD-L1, lead to local regulatory responses capable of abrogating allograft rejection in a model of cell-based transplantation. We herein provide evidence that enhanced immune regulating function can be obtained when presentation of SA-PD-L1 is achieved through a longer more flexible PEG chain. Presentation of SA-PD-L1 through a linker of high molecular weight, and thus longer length (10 kDa, 60 nm in length), led to enhance conversion of naive T cells into T regulatory cells (Tregs) in vitro. In addition, using a subcutaneous implant model and protein tethered through three different linker sizes (6, 30, and 60 nm) to the surface of PEG hydrogels, we demonstrated that longer linkers promoted PD-1 immunomodulatory role in vivo through three main functions: (1) augmenting immune cell recruitment at the transplant site; (2) promoting the accumulation of naive Tregs expressing migratory markers; and (3) dampening CD8+ cytolytic molecule production while augmenting expression of exhaustion phenotypes locally. Notably, accumulation of Treg cells at the implant site persisted for over 30 days postimplantation, an effect not observed when protein was presented with the shorter version of the linkers (6 and 30 nm). Collectively, these studies reveal a facile approach by which PD-L1 function can be modulated through external tuning of synthetic presenting linkers. Impact statement Recently, there has been a growing interest in immune checkpoint molecules as potential targets for tolerance induction, including programmed cell death protein-1 (PD-1). However, how the mechanics of ligand binding to PD-1 receptor affect downstream activation signaling pathways remains unresolved. By taking advantage of the effect of polyethylene glycol chain length on molecule kinetics in an aqueous solution, we herein show that PD-L1 function can be amplified by adjusting the length of the grafting linker. Our results uncover a potential facile mechanism that can be exploited to advance the role of immune checkpoint ligands, in particular PD-L1, in tolerance induction for immunosuppression-free cell-based therapies.

Coordinated inositide lipid-phosphatase activities of synaptojanin modulates actin cytoskeleton organization

Synaptojanin proteins are evolutionarily conserved regulators of vesicle transport and membrane homeostasis. Disruption of synaptojanin function has been implicated in a wide range of neurological disorders. Synaptojanins act as dual-functional lipid phosphatases capable of hydrolyzing a variety of phosphoinositides (PIPs) through autonomous SAC1-like PIP 4-phosphatase and PIP2 5-phosphatase domains. The rarity of an evolutionary configuration of tethering two distinct enzyme activities in a single protein prompted us to investigate their individual and combined roles in budding yeast. Both PIP and PIP2 phosphatase activities are encoded by multiple gene products and are independently essential for cell viability. In contrast, we observed that the synaptojanin proteins utilized both lipid-phosphatase activities to properly coordinate polarized distribution of actin during the cell cycle. Expression of each activity untethered (in trans) failed to properly reconstitute the basal actin regulatory activity; whereas tethering (in cis), even through synthetic linkers, was sufficient to complement these defects. Studies of chimeric proteins harboring PIP and PIP2 phosphatase domains from a variety of gene products indicate synaptojanin proteins have highly specialized activities and that the length of the linker between the lipid-phosphatase domains is critical for actin regulatory activity. Our data are consistent with synaptojanin possessing a strict requirement for both two-domain configuration for some but not all functions and provide mechanistic insights into a coordinated role of tethering distinct lipid-phosphatase activities.

DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

The Use of Mass Spectrometry in Therapeutic Protein Biologics License Applications: A Retrospective Review Revisited.

Biologic license applications (BLAs) for 93 therapeutic proteins approved between 2016 and 2020 were analyzed for use of mass spectrometry (MS) as a follow up to a previous study that assessed MS use in BLAs from 2000 to 2015. Thirty percent of these BLAs were biosimilars, while only one biosimilar BLA was approved prior to 2016. This analysis evaluated the use of a variety of MS techniques and instrumentation. Results were further interpreted based on the relationship of MS use over time, between drug types, and between new drugs and biosimilars. MS data were included in 93 BLAs examined. The top eight quality attributes most assessed by MS in rank order were amino acid sequence, molecular mass, oxidation, disulfide bonds, deamidation, glycosylation, N-terminal sequence variants, and C-terminal sequence variants. These attributes were the same top attributes seen previously from BLAs approved between 2000 and 2015, and the use of MS to analyze them generally continued to increase across the new time frame. The average number of attributes analyzed by MS per BLA also continued to increase over the extended time frame of 21 years. High-resolution, accurate mass instrumentation such as the Orbitrap and time-of-flight (TOF) usage increased over time for all assessed attributes, while matrix-assisted laser desorption/ionization (MALDI)-TOF/(TOF) usage decreased. From highest to lowest rank, the top 11 attributes were antibody drug conjugate (ADC) characterization (i.e., drug load distribution/drug to antibody ratio (DAR), ADC and linkage site, and synthetic linker), isomerization, folding/higher-order structure (HOS), truncation, host cell proteins (HCPs), sequence variants (amino acid substitutions), succinimidation, glycation, PEGylation, charge variants, and oxidation.

Rational Construction of Protein-Mimetic Nano-Switch Systems Based on Secondary Structure Transitions of Synthetic Polypeptides.

The manipulation of the flexibility/rigidity of polymeric chains to control their function is commonly observed in natural macromolecules but largely unexplored in synthetic systems. Herein, we construct a series of protein-mimetic nano-switches consisting of a gold nanoparticle (GNP) core, a synthetic polypeptide linker, and an optically functional molecule (OFM), whose biological function can be dynamically regulated by the flexibility of the polypeptide linker. At the dormant state, the polypeptide adopts a flexible, random-coiled conformation, bringing GNP and OFM in close proximity that leads to the "turn-off" of the OFM. Once treated with alkaline phosphatase (ALP), the nano-switches are activated due to the increased separation distance between GNP and OFM driven by the coil-to-helix and flexible-to-rigid transition of the polypeptide linker. The nano-switches therefore enable selective fluorescence imaging or photodynamic therapy in response to ALP overproduced by tumor cells. The control over polymer flexibility represents an effective strategy to manipulate the optical activity of nano-switches, which mimics the delicate structure-property relationship of natural proteins.

Dual transgene amelioration of Lama2-null muscular dystrophy.

Null mutations of the Lama2-gene cause a severe congenital muscular dystrophy and associated neuropathy. In the absence of laminin-α2 (Lmα2) there is a compensatory replacement by Lmα4, a subunit that lacks the polymerization and α-dystroglycan (αDG)-binding properties of Lmα2. The dystrophic phenotype in the dy3K/dy3K Lama2-/- mouse were evaluated with transgenes driving expression of two synthetic laminin-binding linker proteins. Transgenic muscle-specific expression of αLNNd, a chimeric protein that enables α4-laminin polymerization, and miniagrin (mag), a protein that increases laminin binding to the receptor αDG, separately improved median mouse survival two-fold. The double transgenes (DT) improved mean survival three-fold with increases in overall body weight, muscle size, and grip strength, but, given absence of neuronal expression, did not prevent hindlimb paresis. Muscle improvements included increased myofiber size and number and reduced fibrosis. Myofiber hypertrophy with increased mTOR and Akt phosphorylation were characteristics of mag-dy3K/dy3K and DT-dy3K/dy3K muscle. Elevations of matrix-bound α4-, β1 and γ1 laminin subunits were detected in muscle extracts and immunostained sections in response to DT expression. Collectively, these findings reveal a complimentary polymerization and αDG-binding benefit to Lama2-/- mouse muscle largely mediated through modified laminin-411.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer’s disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

DNA Strand Displacement Driven by Host-Guest Interactions.

Base-pair-driven toehold-mediated strand displacement (BP-TMSD) is a fundamental concept employed for constructing DNA machines and networks with a gamut of applications—from theranostics to computational devices. To broaden the toolbox of dynamic DNA chemistry, herein, we introduce a synthetic surrogate termed host–guest-driven toehold-mediated strand displacement (HG-TMSD) that utilizes bioorthogonal, cucurbit[7]uril (CB[7]) interactions with guest-linked input sequences. Since control of the strand-displacement process is salient, we demonstrate how HG-TMSD can be finely modulated via changes to the structure of the input sequence (including synthetic guest head-group and/or linker length). Further, for a given input sequence, competing small-molecule guests can serve as effective regulators (with fine and coarse control) of HG-TMSD. To show integration into functional devices, we have incorporated HG-TMSD into machines that control enzyme activity and layered reactions that detect specific microRNA.

Endoplasmic reticulum-mitochondria miscommunication is an early and causal trigger of hepatic insulin resistance and steatosis.

Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment. We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations. Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index. ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis. The literature suggests that interactions between the endoplasmic reticulum and mitochondria could play a role in hepatic insulin resistance and steatosis during chronic obesity. In the present study, we reappraised the time-dependent regulation of hepatic endoplasmic reticulum-mitochondria interactions and calcium exchange, investigating reversibility and causality, in mice with diet-induced obesity. We also assessed the relevance of our findings to humans. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis that can be improved by nutritional strategies.

From the Editor’s Desk…

From the Editor’s Desk…

Research Progress of Antibody-drug Conjugates in Advanced Non-small Cell Lung Cancer

肺癌是全球发病率和死亡率最高的恶性肿瘤之一，非小细胞肺癌（non-small cell lung cancer, NSCLC）是肺癌重要的病理类型之一，且晚期患者预后较差，内科治疗仍是其主要治疗手段。抗体偶联药物（antibody-drug conjugates, ADCs）是一类非常有潜力的新型抗肿瘤药物，由单克隆抗体和小分子细胞毒药物通过连接子偶联而成，在肺癌等实体瘤中应用前景广阔。本文对现阶段ADCs在晚期NSCLC中的作用机制和研究进展进行综述。

Linkers: An Assurance for Controlled Delivery of Antibody-Drug Conjugate.

As one of the major therapeutic options for cancer treatment, chemotherapy has limited selectivity against cancer cells. Consequently, this therapeutic strategy offers a small therapeutic window with potentially high toxicity and thus limited efficacy of doses that can be tolerated by patients. Antibody-drug conjugates (ADCs) are an emerging class of anti-cancer therapeutic drugs that can deliver highly cytotoxic molecules directly to cancer cells. To date, twelve ADCs have received market approval, with several others in clinical stages. ADCs have become a powerful class of therapeutic agents in oncology and hematology. ADCs consist of recombinant monoclonal antibodies that are covalently bound to cytotoxic chemicals via synthetic linkers. The linker has a key role in ADC outcomes because its characteristics substantially impact the therapeutic index efficacy and pharmacokinetics of these drugs. Stable linkers and ADCs can maintain antibody concentration in blood circulation, and they do not release the cytotoxic drug before it reaches its target, thus resulting in minimum off-target effects. The linkers used in ADC development can be classified as cleavable and non-cleavable. The former, in turn, can be grouped into three types: hydrazone, disulfide, or peptide linkers. In this review, we highlight the various linkers used in ADC development and their design strategy, release mechanisms, and future perspectives.

Four-Layered Intramolecular Parallel G-Quadruplex with Non-Nucleotide Loops: An Ultra-Stable Self-Folded DNA Nano-Scaffold.

A four-stranded scaffold of nucleic acids termed G-quadruplex (G4) has found growing applications in nano- and biotechnology. Propeller loops are a hallmark of the most stable intramolecular parallel-stranded G4s. To date, propeller loops have been observed to span only a maximum of three G-tetrad layers. Going beyond that would allow creation of more stable scaffolds useful for building robust nanodevices. Here we investigate the formation of propeller loops spanning more than three layers. We show that native nucleotide sequences are incompatible toward this goal, and we report on synthetic non-nucleotide linkers that form a propeller loop across four layers. With the established linkers, we constructed a four-layered intramolecular parallel-stranded G4, which exhibited ultrahigh thermal stability. Control on loop design would augment the toolbox toward engineering of G4-based nanoscaffolds for diverse applications.

Insights into Selectin Inhibitor Design from Endogenous Isomeric Ligands of SLea and SLex.

Selectins interact with cell-surface glycans to promote the initial tethering and rolling of leukocytes, and these interactions are targets for designs of inhibitors to neutralize diseases related to excessive inflammatory responses in many cardiovascular and immune dysfunctions, as well as tumor markers in different cancers. The isomeric endogenous tetrasaccharides, sialyl Lewis X (sLex) and sialyl Lewis A (sLea), are minimal sugar structures required for selectin binding. Understanding their subtle structural variances and significant advanced binding strengths of sLea over sLex could benefit the rational designs for selectin inhibitors. Modeling based on the E-selectin-sLex crystal structure in the present study demonstrated that the N-acetyl group of GlcNAc in sLex could form steric hindrances in the E-selectin-sLex complex, but the hydroxy methylene group of GlcNAc in sLea at the same position allows for stronger binding interactions. The subsequent designed inhibitor with a synthetic accessible linker molecule that has no exo-cyclic moieties replacing GlcNAc displayed comparable dynamic and energetic binding features to sLea. The present study deciphered the clues from endogenous isomeric sLea and sLex and provided insights into designing selectin inhibitors with simplified synthesis.

A Simplistic Single-Step Method for Preparing Biomimetic Nanoparticles from Endogenous Biomaterials.

Many works utilize products isolated from nature as capping agents to functionalize gold nanoparticles for targeting and therapeutic applications. Some of the most advanced of these strategies utilize complex multicomponent biomaterials, such as whole cell-membranes, for nanoparticle functionalization strategies for evading or initializing immune response as well as for targeting. Strategies like these, wherein whole cell membrane is utilized for functionalization, take advantage of the complexity of the protein-lipid content and organization, which cells normally use for communication and interaction (instilling these capacities to nanoparticle vectors). Many approaches for achieving this in functionalizing the surface of nanoparticles rely on multistep processes, which necessitate the addition and then removal of synthetic molecules, heating, or pH modifications. These processes can have deleterious modifying effects on the functionalizing biomolecules, resulting in loss of product and time during each purification step, as well as potentially changing the biomolecule functionality toward a nondesirable effect. Here, we describe methods for forming gold nanoparticles at room temperature in a single step, functionalized with proteins, using nicotinamide adenine dinucleotide (NADH). This process enables formation of nanoparticles that can be functionalized by individual proteins (demonstrated with FBS) or whole cells membrane (extracted from B16F10 cells). This work is derivative from observations found in the literature by us and others, that mammalian cells are capable of producing gold nanoparticles from ionic gold without the supplementation of chemical species. The products of this single-step synthesis described herein have been optimized to maintain biomolecule integrity and so that there are no further purification steps required. To characterize the nanoparticles in terms of their shape, size, surface functionality, and biomolecule integrity throughout development, we employed light-based spectroscopy techniques, molecular modeling, electron microscopy, light scattering, and gel electrophoresis techniques. In order to compare the optimized biomolecule-functionalized nanoparticles against current standards (which require synthetic linkers, heating, or pH manipulation), we employed metabolic and live/dead assays as well as light-based microscopy/spectroscopy in vitro. In comparing our synthetic process against others for forming gold nanoparticles functionalized with complex biomolecule components (whole-cell membrane), we found that this process had superior particle internalization. Our strategy has similar outlets for application to these other works, however, because this process is entirely reliant on endogenous biomaterials and has additional potential.

Floppy linkers direct bacterial proteins

Gram-negative bacteria such as Escherichia coli have a cell envelope consisting of an inner and outer membrane. About one-third of all E. coli proteins are localized in the cell envelope, with some proteins associated with the inner membrane and others with the outer membrane. The process by which this sorting happens is poorly understood. Jean-François Collet of Université catholique de Louvain and coworkers have now found that in E. coli , half the lipoproteins located in the outer membrane have a long, intrinsically disordered linker attached to their N terminus ( Nat. Chem. Biol. 2021, DOI: 10.1038/s41589-021-00845-z ). Both the length and the disorder of the linker help determine where the proteins end up, the researchers find. When synthetic linkers replace the natural ones, the proteins are still routed to the outer membrane if the synthetic linkers are unstructured and have more than 22 amino acids. With linkers that were