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1. Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate

   https://doi.org/10.1007/s00604-023-06068-4  

   Saini, Gunjan; Parasa, Mrugesh Krishna; Clayton, Katherine N.; Fraseur, Julia G.; Bolton, Scott C.; Lin, Kevin P.; Wereley, Steven T.; Kinzer-Ursem, Tamara L. (December 2023, Microchimica Acta)

   Abstract Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins. Graphical Abstract 

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2. Biotin-Conjugated Cellulose Nanofibers Prepared via Copper-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) “Click” Chemistry

   https://doi.org/10.3390/nano10061172  

   Goodge, Katarina; Frey, Margaret (June 2020, Nanomaterials)

   null (Ed.)

   As potential high surface area for selective capture in diagnostic or filtration devices, biotin-cellulose nanofiber membranes were fabricated to demonstrate the potential for specific and bio-orthogonal attachment of biomolecules onto nanofiber surfaces. Cellulose acetate was electrospun and substituted with alkyne groups in either a one- or two-step process. The alkyne reaction, confirmed by FTIR and Raman spectroscopy, was dependent on solvent ratio, time, and temperature. The two-step process maximized alkyne substitution in 10/90 volume per volume ratio (v/v) water to isopropanol at 50 °C after 6 h compared to the one-step process in 80/20 (v/v) at 50 °C after 48 h. Azide-biotin conjugate “clicked” with the alkyne-cellulose via copper-catalyzed alkyne-azide cycloaddition (CuAAC). The biotin-cellulose membranes, characterized by FTIR, SEM, Energy Dispersive X-ray spectroscopy (EDX), and XPS, were used in proof-of-concept assays (HABA (4′-hydroxyazobenzene-2-carboxylic acid) colorimetric assay and fluorescently tagged streptavidin assay) where streptavidin selectively bound to the pendant biotin. The click reaction was specific to alkyne-azide coupling and dependent on pH, ratio of ascorbic acid to copper sulfate, and time. Copper (II) reduction to copper (I) was successful without ascorbic acid, increasing the viability of the click conjugation with biomolecules. The surface-available biotin was dependent on storage medium and time: Decreasing with immersion in water and increasing with storage in air. 

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3. New 1,2,3-Triazoles from (R)-Carvone: Synthesis, DFT Mechanistic Study and In Vitro Cytotoxic Evaluation

   https://doi.org/10.3390/molecules27030769  

   Oubella, Ali; Bimoussa, Abdoullah; N’ait Oussidi, Abdellah; Fawzi, Mourad; Auhmani, Aziz; Morjani, Hamid; Riahi, Abdelkhalek; Esseffar, M’hamed; Parish, Carol; Ait Itto, Moulay Youssef (February 2022, Molecules)

   Aseries of novel 1,4-disubstituted 1,2,3-triazoles were synthesized from an (R)-carvone terminal alkyne derivative via a Cu (I)-catalyzed azide–alkyne cycloaddition reaction using CuSO4,5H2O as the copper (II) source and sodium ascorbate as a reducing agent which reduces Cu (II) into Cu (I). All the newly synthesized 1,2,3-triazoles 9a–h were fully identified on the basis of their HRMS and NMR spectral data and then evaluated for their cell growth inhibition potential by MTS assay against HT-1080 fibrosarcoma, A-549 lung carcinoma, and two breast adenocarcinoma (MCF-7 and MDA-MB-231) cell lines. Compound 9d showed notable cytotoxic effects against the HT-1080 and MCF-7 cells with IC50 values of 25.77 and 27.89 µM, respectively, while compound 9c displayed significant activity against MCF-7 cells with an IC50 value of 25.03 µM. Density functional calculations at the B3LYP/6-31G* level of theory were used to confirm the high reactivity of the terminal alkyne as a dipolarophile. Quantum calculations were also used to investigate the mechanism of both the uncatalyzed and copper (I)-catalyzed azide–alkyne cycloaddition reaction (CuAAC). The catalyzed reaction gives complete regioselectivity via a stepwise mechanism streamlining experimental observations. The calculated free-energy barriers 4.33 kcal/mol and 29.35 kcal/mol for the 1,4- and 1,5-regioisomers, respectively, explain the marked regioselectivity of the CuAAC reaction. 

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4. Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling

   https://doi.org/10.1002/cpz1.497  

   Song, Jiabao; Han, Zhen; Zheng, Y. George (July 2022, Current Protocols)

   Abstract Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization Support Protocol 1: Expression and purification of HAT mutants Support Protocol 2: Synthesis of Ac‐CoA surrogates Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates Basic Protocol 3: Chemoproteomic identification of HAT substrates Basic Protocol 4: Validation of specific HAT substrates with western blotting 

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5. A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling

   https://doi.org/10.1038/s41467-021-26240-9  

   Banerjee, Daipayan; Langberg, Kurt; Abbas, Salar; Odermatt, Eric; Yerramothu, Praveen; Volaric, Martin; Reidenbach, Matthew A.; Krentz, Kathy J.; Rubinstein, C. Dustin; Brautigan, David L.; et al (December 2021, Nature Communications)

   null (Ed.)

   Abstract Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD + ; meanwhile, restoring NAD + levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN ( Crassostrea virginica and Nematostella vectensis ), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity. 

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