JoeyKelly

Huang et al. present a new strategy termed ProLORT that rely on APEX2-catalysed proximity labeling and an orthogonal trap pipeline, to directly decipher the HDAC8-targeted substrates in living cells. ProLORT facilitates the identification of CTTN acetylated site targeted by HDAC8 for the modulation of cell motility.

Enhance immune-killing: A ultra-galactocation to sialic acid strategy is designed by using a penta-functional dendritic probe (Den@5F) to simultaneously block sialic acid and hugely introduce galactose on tumor cells in vivo for the enhancement of immune-killing.

Abstract

Glycans on tumor cell surface have significant impacts in the immune-killing process. Here an ultra-galactocation to sialic acid (Sia) strategy is designed to hugely introduce galactose (Gal) to Sia and on tumor cells in vivo by using a penta-functional dendritic probe (Den@5F), which efficiently enhances the immune-killing of tumor cells. The Den@5F contains five different kinds of functional groups, including Gal, Cy5, amino, phenylboronic acid (PBA) and 4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butanoate (mNB), which can be conveniently prepared through a two-step reaction. After injecting into the tumor-bearing mouse, Den@5F can efficiently block Sia through the specific recognition between PBA and Sia on tumor cells and hugely introduce Gal through the subsequent photo-crosslinking between mNB and amino groups to multiply conjugate excessive Den@5Fs. The comprehensively blocked Sia can prevent the immune escape, and the hugely introduced Gal can promote the immune stimulation of the immune cells, which lead to an efficient enhancement of the immune-killing. The proposed strategy provides a significant and promising tool to promote the clinical immunotherapy of tumor.

Nature Communications, Published online: 28 February 2024; doi:10.1038/s41467-024-45987-5

Anti-CRISPR proteins can help to evade bacterial immunity. Here, the authors elucidate the mechanism of AcrIIA15, which inhibits CRISPR-Cas9 system and also functions as a transcriptional repressor of itself as a fusion protein.

Nature Biotechnology, Published online: 26 February 2024; doi:10.1038/s41587-024-02168-5

Improved linker technologies and broadening payload options mark a coming of age for these precision cancer therapies.

A cascade of three enzymes, E1−E2−E3, is responsible for transferring ubiquitin to target proteins, but the role of the central E2 has been largely overlooked. This work presents a new method, called targeted charging of ubiquitin to E2, or tCUbE, that can follow ubiquitin all the way through its cascade to report on E2 activity and interactions in living mammalian cells.

Abstract

A cascade of three enzymes, E1−E2−E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method—targeted charging of ubiquitin to E2 (tCUbE)—that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2−E3-substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS.

Nature Chemical Biology, Published online: 06 February 2024; doi:10.1038/s41589-023-01539-4

SUGAR-TARGET is a modular platform for the homogeneous synthesis of enzymes with controlled N-linked glycosylation using a one-step immobilization/purification method.

Nature Reviews Drug Discovery, Published online: 05 February 2024; doi:10.1038/d41573-024-00021-7

Macrocyclic peptides thwart Gram-negative bacteria

Su et al. develop a genetically engineered and modular transferrin receptor-lysosome targeting chimera (TfR-LYTAC) for lysosomal degradation of extracellular proteins. Bacterial outer membrane vesicles (OMVs) are used to target TfR-LYTAC in the tumor and the engineered OMV-LYTACs combine cancer immunotherapy of PD-1/PD-L1 inhibition and immune activation through bacterial OMVs.

Splicing Neo Antigen Finder (SNAF) identifies shared immunogenic MHC-presented splicing neoantigens and tumor-specific transmembrane isoforms.

Nature Reviews Drug Discovery, Published online: 15 January 2024; doi:10.1038/d41573-024-00013-7

Circular RNA vaccines expose cryptic peptides

Nature Chemical Biology, Published online: 28 December 2023; doi:10.1038/s41589-023-01505-0

Cyclic peptides can bind challenging disease targets, but their oral application is hindered by digestion and absorption issues. We developed a versatile method for the synthesis and functional screening of vast numbers of synthetic cyclic peptides and identified peptides with high inhibitory activity, stability and oral bioavailability in rats.

Peptide-bismuth bicycles can effectively penetrate human cell membranes. They are easy to synthesize, require only three positive charges for cell entry, and their uptake can be monitored by mass spectrometry due to the unique ability to trace bismuth. This discovery offers an alternative to fluorophore labeling and opens new possibilities for designing therapeutic agents with improved cellular uptake and traceability.

Abstract

Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R₈), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.

Nature Microbiology, Published online: 18 December 2023; doi:10.1038/s41564-023-01544-2

Staphylococcus epidermidis IVK83, a nasal commensal, produces an extremely short-lived, broad-spectrum antimicrobial peptide–polyene called epifadin, which eliminates Staphylococcus aureus in vitro and in vivo.

Cyclic peptide proteasome stimulators: Cyclic peptides inspired by natural products activate ubiquitin independent degradation of disordered proteins. Development and utilization of a flow-based proteasome assay enabled confirmation of the activity of cyclic peptides in cells, making them valuable chemical tools for studying proteasome activation.

Abstract

The proteasome degrades proteins, which is essential for cellular homeostasis. Ubiquitin independent proteolysis degrades highly disordered and misfolded proteins. A decline of proteasomal activity has been associated with multiple neurodegenerative diseases due to the accumulation of misfolded proteins. In this work, cyclic peptide proteasome stimulators (CyPPSs) that enhance the clearance of misfolded proteins were discovered. In the initial screen of predicted natural products (pNPs), several cyclic peptides were found to stimulate the 20S core particle (20S CP). Development of a robust structural activity relationship led to the identification of potent, cell permeable CyPPSs. In vitro assays revealed that CyPPSs stimulate degradation of highly disordered and misfolded proteins without affecting ordered proteins. Furthermore, using a novel flow-based assay for proteasome activity, several CyPPSs were found to stimulate the 20S CP in cellulo. Overall, this work describes the development of CyPPSs as chemical tools capable of stimulating the proteasome and provides strong support for proteasome stimulation as a therapeutic strategy for neurodegenerative diseases.