Selective Protein Degradation through Tetrazine Ligation of Genetically Incorporated Unnatural Amino Acids.

Small molecule-responsive tags for targeted protein degradation are valuable tools for fundamental research and drug target validation. Here, we show that genetically incorporated unnatural amino acids bearing a strained alkene or alkyne functionality can act as a minimalist tag for targeted protein degradation. Specifically, we observed the degradation of strained alkene- or alkyne-containing kinases and E2 ubiquitin-conjugating enzymes upon treatment with hydrophobic tetrazine conjugates. The extent of the induced protein degradation depends on the identity of the target protein, unnatural amino acid, and tetrazine conjugate, as well as the site of the unnatural amino acid in the target protein. Mechanistic studies revealed proteins undergo proteasomal degradation after tetrazine tethering, and the identity of tetrazine conjugates influences the dependence of ubiquitination on protein degradation. This work provides an alternative approach for targeted protein degradation and mechanistic insight, facilitating the future development of more effective targeted protein degradation strategies.

The risk of geriatric syndromes in older COVID-19 survivors among the nonvaccinated population: a real world retrospective cohort study.

BACKGROUND: We aimed to analyse the differences in the risk of geriatric syndromes between older adults with and without coronavirus disease 2019 (COVID-19). METHODS: We conducted a retrospective cohort study of patients from the US Collaborative Network in the TriNetX between January 1, 2020, and December 31, 2022. We included individuals aged older than 65 years with at least 2 health care visits who underwent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) tests during the study period. We excluded those with SARS-CoV-2 vaccination, diagnosis with neoplasm and geriatric syndromes before the index date, and death within 30 days after the index date. The index date was defined as the first date of the PCR test for SARS-CoV-2 during the study period. Hazard ratios (HRs) and 95% confidence intervals (CIs) for eight geriatric syndromes were estimated for propensity score-matched older adults with and without COVID-19. Subgroup analyses of sex and age were also performed. RESULTS: After propensity score matching, 315 826 patients were included (mean [standard deviation] age, 73.5 [6.4] years; 46.7% males and 51.7% females). The three greatest relative increases in the risk of geriatric syndromes in the COVID-19 cohort were cognitive impairment (HR: 3.13; 95% CI: 2.96-3.31), depressive disorder (HR: 2.72; 95% CI: 2.62-2.82) and pressure injury (HR: 2.52; 95% CI: 2.34-2.71). CONCLUSIONS: The risk of developing geriatric syndromes is much higher in the COVID-19 cohort. It is imperative that clinicians endeavour to prevent or minimise the development of these syndromes in the post-COVID-19 era.

Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies.

Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between preclinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine.

Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling.

Recent advances in chemical proteomics have focused on developing chemical probes that react with nucleophilic amino acid residues. Although histidine is an attractive candidate due to its importance in enzymatic catalysis, metal binding and protein-protein interaction, its moderate nucleophilicity poses challenges. Its modification is frequently influenced by cysteine and lysine, which results in poor selectivity and narrow proteome coverage. Here we report a singlet oxygen and chemical probe relay labelling method that achieves high selectivity towards histidine. Libraries of small-molecule photosensitizers and chemical probes were screened to optimize histidine labelling, enabling histidine profiling in live cells with around 7,200 unique sites. Using NMR spectroscopy and X-ray crystallography, we characterized the reaction mechanism and the structures of the resulting products. We then applied this method to discover unannotated histidine sites key to enzymatic activity and metal binding in select metalloproteins. This method also revealed the accessibility change of histidine mediated by protein-protein interaction that influences select protein subcellular localization, underscoring its capability in discovering functional histidines.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

The gut microbiota modulate locomotion via vagus-dependent glucagon-like peptide-1 signaling.

Locomotor activity is an innate behavior that can be triggered by gut-motivated conditions, such as appetite and metabolic condition. Various nutrient-sensing receptors distributed in the vagal terminal in the gut are crucial for signal transduction from the gut to the brain. The levels of gut hormones are closely associated with the colonization status of the gut microbiota, suggesting a complicated interaction among gut bacteria, gut hormones, and the brain. However, the detailed mechanism underlying gut microbiota-mediated endocrine signaling in the modulation of locomotion is still unclear. Herein, we show that broad-spectrum antibiotic cocktail (ABX)-treated mice displayed hypolocomotion and elevated levels of the gut hormone glucagon-like peptide-1 (GLP-1). Blockade of the GLP-1 receptor and subdiaphragmatic vagal transmission rescued the deficient locomotor phenotype in ABX-treated mice. Activation of the GLP-1 receptor and vagal projecting brain regions led to hypolocomotion. Finally, selective antibiotic treatment dramatically increased serum GLP-1 levels and decreased locomotion. Colonizing Lactobacillus reuteri and Bacteroides thetaiotaomicron in microbiota-deficient mice suppressed GLP-1 levels and restored the hypolocomotor phenotype. Our findings identify a mechanism by which specific gut microbes mediate host motor behavior via the enteroendocrine and vagal-dependent neural pathways.

Butterflies in the gut: the interplay between intestinal microbiota and stress.

Psychological stress is a global issue that affects at least one-third of the population worldwide and increases the risk of numerous psychiatric disorders. Accumulating evidence suggests that the gut and its inhabiting microbes may regulate stress and stress-associated behavioral abnormalities. Hence, the objective of this review is to explore the causal relationships between the gut microbiota, stress, and behavior. Dysbiosis of the microbiome after stress exposure indicated microbial adaption to stressors. Strikingly, the hyperactivated stress signaling found in microbiota-deficient rodents can be normalized by microbiota-based treatments, suggesting that gut microbiota can actively modify the stress response. Microbiota can regulate stress response via intestinal glucocorticoids or autonomic nervous system. Several studies suggest that gut bacteria are involved in the direct modulation of steroid synthesis and metabolism. This review provides recent discoveries on the pathways by which gut microbes affect stress signaling and brain circuits and ultimately impact the host's complex behavior.

Microbial metabolites regulate social novelty via CaMKII neurons in the BNST.

Social novelty is a cognitive process that is essential for animals to interact strategically with conspecifics based on their prior experiences. The commensal microbiome in the gut modulates social behavior through various routes, including microbe-derived metabolite signaling. Short-chain fatty acids (SCFAs), metabolites derived from bacterial fermentation in the gastrointestinal tract, have been previously shown to impact host behavior. Herein, we demonstrate that the delivery of SCFAs directly into the brain disrupts social novelty through distinct neuronal populations. We are the first to observe that infusion of SCFAs into the lateral ventricle disrupted social novelty in microbiome-depleted mice without affecting brain inflammatory responses. The deficit in social novelty can be recapitulated by activating calcium/calmodulin-dependent protein kinase II (CaMKII)-labeled neurons in the bed nucleus of the stria terminalis (BNST). Conversely, chemogenetic silencing of the CaMKII-labeled neurons and pharmacological inhibition of fatty acid oxidation in the BNST reversed the SCFAs-induced deficit in social novelty. Our findings suggest that microbial metabolites impact social novelty through a distinct neuron population in the BNST.

Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation.

Site-specific modification of proteins has wide applications in probing and perturbing biological systems. A popular means to achieve such a modification on a target protein is through a reaction between bioorthogonal functionalities. Indeed, various bioorthogonal reactions have been developed, including a recently reported reaction between 1,2-aminothiol and ((alkylthio)(aryl)methylene)malononitrile (TAMM). Here, we describe the procedure that combines genetic code expansion and TAMM condensation for site-specific modification of cellular membrane proteins. The 1,2-aminothiol functionality is introduced through a genetically incorporated noncanonical amino acid to a model membrane protein on mammalian cells. Treatment of the cells with a fluorophore-TAMM conjugate leads to fluorescent labeling of the target protein. This method can be applied to modify different membrane proteins on live mammalian cells.

Selective Inhibition of Kinase Activity in Mammalian Cells by Bioorthogonal Ligand Tethering.

Enzymes are critical for cellular functions, and malfunction of enzymes is closely related to many human diseases. Inhibition studies can help in deciphering the physiological roles of enzymes and guide conventional drug development programs. In particular, chemogenetic approaches enabling rapid and selective inhibition of enzymes in mammalian cells have unique advantages. Here, we describe the procedure for rapid and selective inhibition of a kinase in mammalian cells by bioorthogonal ligand tethering (iBOLT). Briefly, a non-canonical amino acid bearing a bioorthogonal group is genetically incorporated into the target kinase by genetic code expansion. The sensitized kinase can react with a conjugate containing a complementary biorthogonal group linked with a known inhibitory ligand. As a result, tethering of the conjugate to the target kinase allows selective inhibition of protein function. Here, we demonstrate this method by using cAMP-dependent protein kinase catalytic subunit alpha (PKA-Cα) as the model enzyme. The method should be applicable to other kinases, enabling their rapid and selective inhibition.

Joint predictability of physical frailty/pre-frailty and subjective memory complaints on mortality risk among cognitively unimpaired older adults.

The aim of the present study was to investigate how frailty/pre-frailty in combination with subjective memory complaints predicts all-cause mortality in community dwelling cognitively unimpaired older adults. There were 1904 community-dwelling cognitively unimpaired persons aged 65 years or older who participated in the 2013 Taiwan National Health Interview Survey with a 5-year follow-up. Frailty was determined based on the fatigue, resistance, ambulation, illness, and loss of weight (FRAIL) scale. Two questions ("Do you have difficulties with your memory or attention?" and "Do you have difficulties with your memory only or attention only or both?") were used to screen for subjective memory complaints (SMC). In this study, 11.9% of participants had both frailty/pre-frailty and SMC. A total of 239 deaths were recorded after 9009.5 person-years of follow-up. After adjustment for other factors, compared with participants who were physically robust with no SMC, participants who reported either SMC alone (HR = 0.88, 95% CI = 0.60-1.27) or were frail/pre-frail alone (HR = 1.32, 95% CI = 0.90-1.92) had no significantly increased mortality risk. However, coexisting frailty/pre-frailty and SMC was associated with a significantly increased hazard ratio for mortality of 1.48 (95% CI = [1.02-2.16]). Our results highlight the high prevalence of co-occurring frailty/pre-frailty and SMC and that this co-occurrence is associated with an increased risk of mortality among cognitively unimpaired older adults.

The deficiency of poly-ß-1,6-N-acetyl-glucosamine deacetylase trigger A. baumannii to convert to biofilm-independent colistin-tolerant cells.

Acinetobacter baumannii is a nosocomial pathogen that can be resistant to antibiotics by rapidly modulating its anti-drug mechanisms. The multidrug-resistant A. baumannii has been considered one of the most threatening pathogens to our society. Biofilm formation and persistent cells within the biofilm matrix are recognized as intractable problems, especially in hospital-acquired infections. Poly-ß-1,6-N-acetyl-glucosamine (PNAG) is one of the important building blocks in A. baumannii's biofilm. Here, we discover a protein phosphoryl-regulation on PNAG deacetylase, AbPgaB1, in which residue Ser411 was phosphorylated. The phosphoryl-regulation on AbPgaB1 modulates the product turnover rate in which deacetylated PNAG is produced and reflected in biofilm production. We further uncovered the PgaB deficient A. baumannii strain shows the lowest level of biofilm production but has a high minimal inhibition concentration to antibiotic colistin and tetracycline. Based on bactericidal post-antibiotic effects and time-dependent killing assays with antibacterial drugs, we claim that the PgaB-deficient A. baumannii converts to colistin-tolerant cells. This study utilizes a biofilm-independent colistin-tolerant model of A. baumannii to further investigate its characteristics and mechanisms to better understand clinical outcomes.

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins.

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(II), as a linker for C-to-N SPCL.

Effect of Trimethine Cyanine Dye- and Folate-Conjugation on the In Vitro Biological Activity of Proapoptotic Peptides.

Despite continuous advances, anticancer therapy still faces several technical hurdles, such as selectivity on cellular and subcellular targets of therapeutics. Toward addressing these limitations, we have combined the use of proapoptotic peptides, trimethine cyanine dye, and folate to target the mitochondria of tumor cells. A series of proapoptotic peptides and their conjugates with a cyanine dye and/or folate were synthesized in the solid phase, and their toxicity in different human cell lines was assessed. Cyanine-bearing conjugates were found to be up to 100-fold more cytotoxic than the parent peptides and to localize in mitochondria. However, the addition of a folate motif did not enhance the potency or selectivity of the resulting conjugates toward tumor cells that overexpress folate receptor α. Furthermore, while dual-labeled constructs were also found to localize within the target organelle, they were not generally selective towards folate receptor α-positive cell lines in vitro.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering.

A general approach for the rapid and selective inhibition of enzymes in cells using a common tool compound would be of great value for research and therapeutic development. We previously reported a chemogenetic strategy that addresses this challenge for kinases, relying on bioorthogonal tethering of a pan inhibitor to a target kinase through a genetically encoded non-canonical amino acid. However, pan inhibitors are not available for many enzyme classes. Here, we expand the scope of the chemogenetic strategy to cysteine-dependent enzymes by bioorthogonal tethering of electrophilic warheads. For proof of concept, selective inhibition of two E2 ubiquitin-conjugating enzymes, UBE2L3 and UBE2D1, was demonstrated in biochemical assays. Further development and optimization of this approach should enable its use in cells as well as with other cysteine-dependent enzymes, facilitating the investigation of their cellular function and validation as therapeutic targets.

Applications of Genetic Code Expansion in Studying Protein Post-translational Modification.

Various post-translational modifications can naturally occur on proteins, regulating the activity, subcellular localization, interaction, or stability of the proteins. However, it can be challenging to decipher the biological implication or physiological roles of site-specific modifications due to their dynamic and sub-stoichiometric nature. Genetic code expansion method, relying on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, enables site-specific incorporation of non-canonical amino acids. Here we focus on the application of genetic code expansion to study site-specific protein post-translational modification in vitro and in vivo. After a brief introduction, we discuss possibilities of incorporating non-canonical amino acids containing post-translational modifications or their mimics into target proteins. This approach is applicable for Ser/Thr/Tyr phosphorylation, Tyr sulfation/nitration/hydroxylation, Lys acetylation/acylation, Lys/His mono-methylation, as well as Arg citrullination. The next section describes the use of a precursor non-canonical amino acid followed by chemical and/or enzymatic reactions to afford the desired modification, such as Cys/Lys acylation, ubiquitin and ubiquitin-like modifications, as well as Lys/Gln methylation. We also discuss means for functional regulation of enzymes involving in post-translational modifications through genetically incorporated non-canonical amino acids. Lastly, the limitations and perspectives of genetic code expansion in studying protein post-translational modification are described.

Quantitative Subcellular Analysis of Cyclic Cell-Penetrating Peptide EJP18 in Nonadherent Cells.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.

IL-33/ST2 Axis Plays a Protective Effect in Streptococcus pyogenes Infection through Strengthening of the Innate Immunity.

Group A Streptococcus (GAS) causes invasive human diseases with the cytokine storm. Interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) axis is known to drive TH2 response, while its effect on GAS infection is unclear. We used an air pouch model to examine the effect of the IL-33/ST2 axis on GAS-induced necrotizing fasciitis. GAS infection induced IL-33 expression in wild-type (WT) C57BL/6 mice, whereas the IL-33- and ST2-knockout mice had higher mortality rates, more severe skin lesions and higher bacterial loads in the air pouches than those of WT mice after infection. Surveys of infiltrating cells in the air pouch of GAS-infected mice at the early stage found that the number and cell viability of infiltrating cells in both gene knockout mice were lower than those of WT mice. The predominant effector cells in GAS-infected air pouches were neutrophils. Absence of the IL-33/ST2 axis enhanced the expression of inflammatory cytokines, but not TH1 or TH2 cytokines, in the air pouch after infection. Using in vitro assays, we found that the IL-33/ST2 axis not only enhanced neutrophil migration but also strengthened the bactericidal activity of both sera and neutrophils. These results suggest that the IL-33/ST2 axis provided the protective effect on GAS infection through enhancing the innate immunity.