Sortase A-Based Post-translational Modifications on Encapsulin Nanocompartments.

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the fields of biotechnology and nanomedicine. In this investigation, we effectively developed a sortase A-mediated protein ligation system in Escherichia coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles' exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research developed an alternative enzymatic ligation method for engineering encapsulin nanoparticles to facilitate the conjugation process.

Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.

Synthesis, Antimicrobial Activities, and Molecular Modeling Studies of Agents for the Sortase A Enzyme.

Sortase A (SrtA) is an attractive target for developing new anti-infective drugs that aim to interfere with essential virulence mechanisms, such as adhesion to host cells and biofilm formation. Herein, twenty hydroxy, nitro, bromo, fluoro, and methoxy substituted chalcone compounds were synthesized, antimicrobial activities and molecular modeling strategies against the SrtA enzyme were investigated. The most active compounds were found to be T2, T4, and T19 against Streptococcus mutans (S. mutans) with MIC values of 1.93, 3.8, 3.94 µg/mL, and docking scores of -6.46, -6.63, -6.73 kcal/mol, respectively. Also, these three active compounds showed better activity than the chlorohexidine (CHX) (MIC value: 4.88 µg/mL, docking score: -6.29 kcal/mol) in both in vitro and in silico. Structural stability and binding free energy analysis of S.mutans SrtA with active compounds were measured by molecular dynamic (MD) simulations throughout 100 nanoseconds (ns) time. It was observed that the stability of the critical interactions between these compounds and the target enzyme was preserved. To prove further, in vivo biological evaluation studies could be conducted for the most promising precursor compounds T2, T4, and T19, and it might open new avenues to the discovery of more potent SrtA inhibitors.

Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling.

BACKGROUND: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems-the ubiquitin proteasome system (UPS) and macroautophagy-mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless, how Ate1 operates through this intricate mechanism remains elusive. METHODS: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofluorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform were used as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofluorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy. RESULTS: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. CONCLUSIONS: Our findings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Global Analysis of Post-Translational Side-Chain Arginylation Using Pan-Arginylation Antibodies.

Arginylation is a post-translational modification mediated by the arginyltransferase 1 (ATE1), which transfers the amino acid arginine to a protein or peptide substrate from a tRNA molecule. Initially, arginylation was thought to occur only on N-terminally exposed acidic residues, and its function was thought to be limited to targeting proteins for degradation. However, more recent data have shown that ATE1 can arginylate side chains of internal acidic residues in a protein without necessarily affecting metabolic stability. This greatly expands the potential targets and functions of arginylation, but tools for studying this process have remained limited. Here, we report the first global screen specifically for side-chain arginylation. We generate and validate "pan-arginylation" antibodies, which are designed to detect side-chain arginylation in any amino acid sequence context. We use these antibodies for immunoaffinity enrichment of side-chain arginylated proteins from wildtype and Ate1 knockout cell lysates. In this way, we identify a limited set of proteins that likely undergo ATE1-dependent side-chain arginylation and that are enriched in specific cellular roles, including translation, splicing, and the cytoskeleton.

Steric-Deficient Oligoglycine Surrogates Facilitate Multivalent and Bifunctional Nanobody Synthesis via Combined Sortase A Transpeptidation and Click Chemistry.

Conferring multifunctional properties to proteins via enzymatic approaches has greatly facilitated recent progress in protein nanotechnology. In this regard, sortase (Srt) A transpeptidation has facilitated many of these developments due to its exceptional specificity, mild reaction conditions, and complementation with other bioorthogonal techniques, such as click chemistry. In most of these developments, Srt A is used to seamlessly tether oligoglycine-containing molecules to a protein of interest that is equipped with the enzyme's recognition sequence, LPXTG. However, the dependence on oligoglycine attacking nucleophiles and the associated cost of certain derivatives (e.g., cyclooctyne) limit the utility of this approach to lab-scale applications only. Thus, the quest to identify appropriate alternatives and understand their effectiveness remains an important area of research. This study identifies that steric and nucleophilicity-associated effects influence Srt A transpeptidation when two oligoglycine surrogates were examined. The approach was further used in complementation with click chemistry to synthesize bivalent and bifunctional nanobody conjugates for application in epithelial growth factor receptor targeting. The overall technique and tools developed here may facilitate the advancement of future nanotechnologies.

Chemically Synthetic d-Sortase Enables Enzymatic Ligation of d-Peptides.

We have described the chemical synthesis of d-Sortase A in large quantity and high purity by a hydrazide ligation strategy. The d-Sortase was fully active toward d-peptides and D/L hybrid proteins, and the ligation efficiency was unaffected by the chirality of the C-terminus substrate. This study points toward using d-sortase ligation as a modern ligation method for d-proteins and D/L hybrid proteins and expands the chemical protein synthesis toolbox in biotechnology.

Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions.

Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.

Phytochelatins: Sulfur-Containing Metal(loid)-Chelating Ligands in Plants.

Phytochelatins (PCs) are small cysteine-rich peptides capable of binding metal(loid)s via SH-groups. Although the biosynthesis of PCs can be induced in vivo by various metal(loid)s, PCs are mainly involved in the detoxification of cadmium and arsenic (III), as well as mercury, zinc, lead, and copper ions, which have high affinities for S-containing ligands. The present review provides a comprehensive account of the recent data on PC biosynthesis, structure, and role in metal(loid) transport and sequestration in the vacuoles of plant cells. A comparative analysis of PC accumulation in hyperaccumulator plants, which accumulate metal(loid)s in their shoots, and in the excluders, which accumulate metal(loid)s in their roots, investigates the question of whether the endogenous PC concentration determines a plant's tolerance to metal(loid)s. Summarizing the available data, it can be concluded that PCs are not involved in metal(loid) hyperaccumulation machinery, though they play a key role in metal(loid) homeostasis. Unraveling the physiological role of metal(loid)-binding ligands is a fundamental problem of modern molecular biology, plant physiology, ionomics, and toxicology, and is important for the development of technologies used in phytoremediation, biofortification, and phytomining.

Harnessing sortase A transpeptidation for advanced targeted therapeutics and vaccine engineering.

The engineering of potent prophylactic and therapeutic complexes has always required careful protein modification techniques with seamless capabilities. In this light, methods that favor unobstructed multivalent targeting and correct antigen presentations remain essential and very demanding. Sortase A (SrtA) transpeptidation has exhibited these attributes in various settings over the years. However, its applications for engineering avidity-inspired therapeutics and potent vaccines have yet to be significantly noticed, especially in this era where active targeting and multivalent nanomedications are in great demand. This review briefly presents the SrtA enzyme and its associated transpeptidation activity and describes interesting sortase-mediated protein engineering and chemistry approaches for achieving multivalent therapeutic and antigenic responses. The review further highlights advanced applications in targeted delivery systems, multivalent therapeutics, adoptive cellular therapy, and vaccine engineering. These innovations show the potential of sortase-mediated techniques in facilitating the development of simple plug-and-play nanomedicine technologies against recalcitrant diseases and pandemics such as cancer and viral infections.

GPApred: The first computational predictor for identifying proteins with LPXTG-like motif using sequence-based optimal features.

The cell surface proteins of gram-positive bacteria are involved in many important biological functions, including the infection of host cells. Owing to their virulent nature, these proteins are also considered strong candidates for potential drug or vaccine targets. Among the various cell surface proteins of gram-positive bacteria, LPXTG-like proteins form a major class. These proteins have a highly conserved C-terminal cell wall sorting signal, which consists of an LPXTG sequence motif, a hydrophobic domain, and a positively charged tail. These surface proteins are targeted to the cell envelope by a sortase enzyme via transpeptidation. A variety of LPXTG-like proteins have been experimentally characterized; however, their number in public databases has increased owing to extensive bacterial genome sequencing without proper annotation. In the absence of experimental characterization, identifying and annotating these sequences is extremely challenging. Therefore, in this study, we developed the first machine learning-based predictor called GPApred, which can identify LPXTG-like proteins from their primary sequences. Using a newly constructed benchmark dataset, we explored different classifiers and five feature encodings and their hybrids. Optimal features were derived using the recursive feature elimination method, and these features were then trained using a support vector machine algorithm. The performance of different models was evaluated using independent datasets, and a final model (GPApred) was selected based on consistency during cross-validation and independent assessment. GPApred can be an effective tool for predicting LPXTG-like sequences and can be further employed for functional characterization or drug targeting. Availability: https://procarb.org/gpapred/.

Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis.

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct contact between macrophages and senescent cells. SCES involved augmented senescent cell expression of CD47 coinciding with increased CD47-modifying enzymes QPCT/L. SCES was reversible by interfering with the SIRPα-CD47-SHP-1 axis or QPCT/L activity. While CD47 expression increased in human and mouse senescent cells in vitro and in vivo, another ITIM-containing protein, CD24, contributed to SCES specifically in human epithelial senescent cells where it compensated for genetic deficiency in CD47. Thus, CD47 and CD24 link the pathogenic effects of senescent cells to homeostatic macrophage functions, such as efferocytosis, which we hypothesize must occur efficiently to maintain tissue homeostasis.

Effects of Phytochelatin-like Gene on the Resistance and Enrichment of Cd2+ in Tobacco.

Phytochelatins (PCs) are class III metallothioneins in plants. They are low molecular-weight polypeptides rich in cysteine residues which can bind to metal ions and affect the physiological metabolism in plants. Unlike other types of metallothioneins, PCs are not the product of gene coding but are synthesized by phytochelatin synthase (PCS) based on glutathione (GSH). The chemical formula of phytochelatin is a mixture of (γ-Glu-Cys)n-Gly (n = 2-11) and is influenced by many factors during synthesis. Phytochelatin-like (PCL) is a gene-encoded peptide (Met-(α-Glu-Cys)11-Gly) designed by our laboratory whose amino acid sequence mimics that of a natural phytochelatin. This study investigated how PCL expression in transgenic plants affects resistance to Cd and Cd accumulation. Under Cd2+ stress, transgenic plants were proven to perform significantly better than the wild-type (WT), regarding morphological traits and antioxidant abilities, but accumulated Cd to higher levels, notably in the roots. Fluorescence microscopy showed that PCL localized in the cytoplasm and nucleus.

Upregulation of Glutaminyl Cyclase Contributes to ERS-Induced Apoptosis in PC12 Cells.

Glutaminyl cyclase (QC) is responsible for converting the N-terminal glutaminyl and glutamyl of the proteins into pyroglutamate (pE) through cyclization. It has been confirmed that QC catalyzes the formation of neurotoxic pE-modified Aß in the brain of AD patients. But the effects of upregulated QC in diverse diseases have not been much clear until recently. Here, RNA sequencing was applied to identify differentially expressed genes (DEGs) in PC12 cells with QC overexpressing or knockdown. A total of 697 DEGs were identified in QC overexpressing cells while only 77 in QC knockdown cells. Multiple bioinformatic approaches revealed that the DEGs in QC overexpressing group were enriched in endoplasmic reticulum stress (ERS) related signaling pathways. The gene expression patterns of 23 DEGs were confirmed by RT-qPCR, in which the genes related to ERS showed the highest consistency. We also revealed the protein levels of GRP78, PERK, CHOP, and PARP-1, and caspase family was significantly upregulated by overexpressing QC. Moreover, overexpressing QC significantly increased apoptosis of PC12 cells in a time dependent manner. However, no significant alteration was observed in QC knockdown cells. Therefore, our study indicated that upregulated QC could induce ERS and apoptosis, which consequently trigger diseases by catalyzing the generation of pE-modified mediators.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition.

The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal-containing protein to the peptidoglycan precursor lipid II (LII). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl-enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

Glutaminyl cyclases, the potential targets of cancer and neurodegenerative diseases.

Glutaminyl cyclases (QC) catalyze the cyclization of proteins and turn N-terminal glutamine or glutamic acid into N-terminal pyroglutamate, resulting in protection of proteins from aminopeptidases and an increase of their stabilities. The aberrant N-terminal pyroglutamate has been found in various diseases, including Alzheimer's disease (AD), Huntington's disease (HD) and cancer. Two kinds of human QC, the secretory sQC and the Golgi resident gQC, are identified to date. Several substrates of sQC involving beta amyloid (Aß), Huntington (HTT) protein and certain inflammatory mediators such as CCL2 and CX3CL1 have been observed to associate with neurodegenerative diseases and cancers. The Golgi resident gQC can modify N-terminus of CD47 that directly influences the interaction of CD47 and SIRPα resulting in the modulations of the immunological surveillance related mechanisms in cancer. Additionally, inflammatory chemokines CCL2 and CX3CL1 can also be modified by gQC. Several QC inhibitors with differential scaffold structures have been developed and investigated. Among these QC inhibitors, PQ912, a benzimidazole-based inhibitor, has been studied in a phase II clinical trial to treat AD. In this review, we will summarize the current knowledge about QCs' tissue expression patterns, their potential cellular substrates in the context of cancers, AD and HD. After introducing QCs' molecular structures and catalysis mechanisms, the structures and efficacies of the currently reported QCs' inhibitors will also be summarized.

Cyanidin chloride protects mice from methicillin-resistant Staphylococcus aureus-induced pneumonia by targeting Sortase A.

Methicillin-resistant Staphylococcus aureus (MRSA) has been developing rapidly in recent years. It poses a severe peril to global health care, and the new strategies to against the MRSA is urgently needed. Sortase A (SrtA) regulates the anchoring of many surface proteins. Compounds repress Staphylococcus aureus (S. aureus) cysteine transpeptidase SrtA are considered adequate potent virulence inhibitors. Then, we describe the identification of an effective SrtA inhibitor, cyanidin chloride, a bioflavonoid compound isolated from various plants. It has a reversible inhibitory effect on SrtA activity at an IC50 of 21.91 µg/mL. As a SrtA inhibitor, cyanidin chloride antagonizes SrtA-related virulence phenotypes due to its breadth and specificity, including fibrinogen adhesion, A549 cell invasion, biofilm formation, and surface protein (SpA) anchoring. Subsequently, molecular docking and fluorescence quenching revealed that SrtA and cyanidin chloride had robust mutual affinity. Further mechanistic studies revealed that Arg-197, Gly-167, and Sep-116 were the key-binding sites mediating the interaction between SrtA and cyanidin chloride. Notably, a significant therapeutic effect of cyanidin chloride in vivo was also observed on the mouse pneumonia model induced by MRSA. In conclusion, our study indicates that cyanidin chloride potentially represents a new candidate SrtA inhibitor for S. aureus and potentially be developed as a new antivirulence agent.

Chemoenzymatic Labeling to Visualize Intercellular Contacts Using Lipidated SortaseA.

Methods to label intercellular contact have attracted attention because of their potential in cell biological and medical applications for the analysis of intercellular communications. In this study, a simple and versatile method for chemoenzymatic labeling of intercellularly contacting cells is demonstrated using a cell-surface anchoring reagent of a poly(ethylene glycol)(PEG)-lipid conjugate. The surface of each cell in the cell pairs of interest were decorated with sortase A (SrtA) and triglycine peptide that were lipidated with PEG-lipid. In the mixture of the two-cell populations, the triglycine-modified cells were enzymatically labeled with a fluorescent labeling reagent when in contact with SrtA-modified cells on a substrate. The selective labeling of the contacting cells was confirmed by confocal microscopy. The method is a promising tool for selective visualization of intercellularly contacting cells in cell mixtures for cell-cell communication analysis.