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.

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase.

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.

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.

The Bacteroidetes Q-rule and glutaminyl cyclase activity increase the stability of extracytoplasmic proteins.

IMPORTANCE: Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase have N-terminal glutamine converted to pyroglutamate. The reaction is catalyzed by the periplasmic enzyme glutaminyl cyclase (QC), which is essential for the growth of Porphyromonas gingivalis and other periodontopathogens. Apparently, pyroglutamyl formation stabilizes extracytoplasmic proteins and/or protects them from proteolytic degradation in the periplasm. Given the role of P. gingivalis as the keystone pathogen in periodontitis, P. gingivalis QC is a promising target for the development of drugs to treat and/or prevent this highly prevalent chronic inflammatory disease leading to tooth loss and associated with severe systemic diseases.

A dual biomarker-targeting probe enables signal-on surface labeling of Staphylococcus aureus.

Imaging or killing of a specific pathogen is of significance for precise therapy. Staphylococcus aureus (S. aureus) is an infectious gram-positive bacteria relying on Sortase A (SrtA) to anchor cell surface protein on peptidoglycan. We herein report signal-on labeling of S. aureus with self-quenched optical probes featuring vancomycin-conjugated SrtA substrate that is flanked by a dabcyl moiety paired with either fluorescein or eosine photosensizer (PS). SrtA-mediated cleavage of the substrate motif releases the dabcyl quencher, leading to covalent labeling of peptidoglycan with fluorescein or PS of restored photophysical property. The dual biomarked-enabled peptidoglycan labeling enables signal-on imaging and effective photodynamic destruction of S. aureus, suggesting a protheranostic approch activatable to SrtA-positive bacteria engaged in myriad diseases.

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.

Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles.

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles.

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV-vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

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.

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.

The preparation of recombinant arginyltransferase 1 (ATE1) for biophysical characterization.

Arginyltransferases (ATE1s) are eukaryotic enzymes that catalyze the non-ribosomal, post-translational addition of the amino acid arginine to an acceptor protein. While understudied, post-translation arginylation and ATE1 have major impacts on eukaryotic cellular homeostasis through both degradative and non-degradative effects on the intracellular proteome. Consequently, ATE1-catalyzed arginylation impacts major eukaryotic biological processes including the stress response, cellular motility, cardiovascular maturation, and even neurological function. Despite this importance, there is a lack of information on the structural and biophysical characteristics of ATE1, prohibiting a comprehensive understanding of the mechanism of this post-translational modification, and hampering efforts to design ATE1-specific therapeutics. To that end, this chapter details a protocol designed for the expression and the purification of ATE1 from Saccharomyces cerevisiae, although the approaches described herein should be generally applicable to other eukaryotic ATE1s. The detailed procedures afford high amounts of pure, homogeneous, monodisperse ATE1 suitable for downstream biophysical analyses such as X-ray crystallography, small angle X-ray scattering (SAXS), and cryo-EM techniques.

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/.

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis.

Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.

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.