Computational Methods for Molecular Understanding of the Antibiotic-Aminoacyl tRNA Synthetase Interaction.

Developing an understanding of the interactions between an antibiotic and its binding site in a pathogen cell is the key to antibiotic design-an important cost-saving methodology compared to the costly and time-consuming random trial-and-error approach. The rapid development of antibiotic resistance provides an impetus for such studies. Recent years have witnessed the beginning of the use of combined computational techniques, including computer simulations and quantum mechanical computations, to understand how antibiotics bind at the active site of aminoacyl tRNA synthetases (aaRSs) from pathogens. Such computational protocols assist the knowledge-based design of antibiotics targeting aaRSs, which are their validated targets. After the ideas behind the protocols and their strategic planning are discussed, the protocols are described along with their major outcomes. This is followed by an integration of results from the different basic protocols. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Analysis of active-site residues from primary sequence of synthetase and transfer RNAs Basic Protocol 2: Molecular dynamics simulation-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex Basic Protocol 3: Quantum mechanical method-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex.

The binding mode of orphan glycyl-tRNA synthetase with tRNA supports the synthetase classification and reveals large domain movements.

As a class of essential enzymes in protein translation, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are organized into two classes of 10 enzymes each, based on two conserved active site architectures. The (αß)2 glycyl-tRNA synthetase (GlyRS) in many bacteria is an orphan aaRS whose sequence and unprecedented X-shaped structure are distinct from those of all other aaRSs, including many other bacterial and all eukaryotic GlyRSs. Here, we report a cocrystal structure to elucidate how the orphan GlyRS kingdom specifically recognizes its substrate tRNA. This structure is sharply different from those of other aaRS-tRNA complexes but conforms to the clash-free, cross-class aaRS-tRNA docking found with conventional structures and reinforces the class-reconstruction paradigm. In addition, noteworthy, the X shape of orphan GlyRS is condensed with the largest known spatial rearrangement needed by aaRSs to capture tRNAs, which suggests potential nonactive site targets for aaRS-directed antibiotics, instead of less differentiated hard-to-drug active site locations.

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates.

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells.

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.

Update of the Pyrrolysyl-tRNA Synthetase/tRNAPyl Pair and Derivatives for Genetic Code Expansion.

The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNAPyl. Owing to the high tolerance of PylRS toward diverse amino acid substrates and great orthogonality in various model organisms, the PylRS/tRNAPyl-derived pairs are ideal for genetic code expansion to insert noncanonical amino acids (ncAAs) into proteins of interest. Since the discovery of cellular components involved in the biosynthesis and genetic encoding of Pyl, synthetic biologists have been enthusiastic about engineering PylRS/tRNAPyl-derived pairs to rewrite the genetic code of living cells. Recently, considerable progress has been made in understanding the molecular phylogeny, biochemical properties, and structural features of the PylRS/tRNAPyl pair, guiding its further engineering and optimization. In this review, we cover the basic and updated knowledge of the PylRS/tRNAPyl pair's unique characteristics that make it an outstanding tool for reprogramming the genetic code. In addition, we summarize the recent efforts to create efficient and (mutually) orthogonal PylRS/tRNAPyl-derived pairs for incorporation of diverse ncAAs by genome mining, rational design, and advanced directed evolution methods.

Chemically Acylated tRNAs are Functional in Zebrafish Embryos.

Genetic code expansion has pushed protein chemistry past the canonical 22 amino acids. The key enzymes that make this possible are engineered aminoacyl tRNA synthetases. However, as the number of genetically encoded amino acids has increased over the years, obvious limits in the type and size of novel side chains that can be accommodated by the synthetase enzyme become apparent. Here, we show that chemically acylating tRNAs allow for robust, site-specific incorporation of unnatural amino acids into proteins in zebrafish embryos, an important model organism for human health and development. We apply this approach to incorporate a unique photocaged histidine analogue for which synthetase engineering efforts have failed. Additionally, we demonstrate optical control over different enzymes in live embryos by installing photocaged histidine into their active sites.

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

Tyrosine-targeted covalent inhibition of a tRNA synthetase aided by zinc ion.

Aminoacyl-tRNA synthetases (AARSs), a family of essential protein synthesis enzymes, are attractive targets for drug development. Although several different types of AARS inhibitors have been identified, AARS covalent inhibitors have not been reported. Here we present five unusual crystal structures showing that threonyl-tRNA synthetase (ThrRS) is covalently inhibited by a natural product, obafluorin (OB). The residue forming a covalent bond with OB is a tyrosine in ThrRS active center, which is not commonly modified by covalent inhibitors. The two hydroxyl groups on the o-diphenol moiety of OB form two coordination bonds with the conserved zinc ion in the active center of ThrRS. Therefore, the ß-lactone structure of OB can undergo ester exchange reaction with the phenolic group of the adjacent tyrosine to form a covalent bond between the compound and the enzyme, and allow its nitrobenzene structure to occupy the binding site of tRNA. In addition, when this tyrosine was replaced by a lysine or even a weakly nucleophilic arginine, similar bonds could also be formed. Our report of the mechanism of a class of AARS covalent inhibitor targeting multiple amino acid residues could facilitate approaches to drug discovery for cancer and infectious diseases.

A standalone editing protein deacylates mischarged canavanyl-tRNAArg to prevent canavanine incorporation into proteins.

Error-free translation of the genetic code into proteins is vitally important for all organisms. Therefore, it is crucial that the correct amino acids are loaded onto their corresponding tRNAs. This process is highly challenging when aminoacyl-tRNA-synthetases encounter structural analogues to the native substrate like the arginine antimetabolite canavanine. To circumvent deleterious incorporation due to tRNA mischarging, editing mechanisms have evolved. However, only for half of the tRNA synthetases, editing activity is known and only few specific standalone editing proteins have been described. Understanding the diverse mechanisms resulting in error-free protein synthesis is of great importance. Here, we report the discovery of a protein that is upregulated upon canavanine stimulation in bacteria that live associated with canavanine-producing plants. We demonstrate that it acts as standalone editing protein specifically deacylating canavanylated tRNAArg. We therefore propose canavanyl-tRNAArgdeacylase (CtdA) as systematic name. Knockout strains show severe growth defects in canavanine-containing media and incorporate high amounts of canavanine into the proteome. CtdA is frequently found under control of guanidine riboswitches, revealing a functional connection of canavanine and guanidine metabolisms. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Error-free translation is one of the most vital processes in all living organisms, but can be substantially challenged by compounds that mimic amino acids. Canavanine, or 5-oxa-arginine, is used as an antimetabolite by higher plants that is toxic due to its incorporation into proteins. We report the discovery of a standalone editing protein specifically deacylating canavanylated tRNAArg that enables the legume rhizosphere inhabitant Pseudomonas canavaninivorans to prevent canavanine mis-incorporation into its proteome. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase.

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development.

Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.

Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells.

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus.

The Role of Nuclear-Encoded Mitochondrial tRNA Charging Enzymes in Human Inherited Disease.

Aminoacyl-tRNA synthetases (ARSs) are highly conserved essential enzymes that charge tRNA with cognate amino acids-the first step of protein synthesis. Of the 37 nuclear-encoded human ARS genes, 17 encode enzymes are exclusively targeted to the mitochondria (mt-ARSs). Mutations in nuclear mt-ARS genes are associated with rare, recessive human diseases with a broad range of clinical phenotypes. While the hypothesized disease mechanism is a loss-of-function effect, there is significant clinical heterogeneity among patients that have mutations in different mt-ARS genes and also among patients that have mutations in the same mt-ARS gene. This observation suggests that additional factors are involved in disease etiology. In this review, we present our current understanding of diseases caused by mutations in the genes encoding mt-ARSs and propose explanations for the observed clinical heterogeneity.

RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.

Mitochondrial RNA metabolism is suggested to occur in identified compartmentalized foci, i.e. mitochondrial RNA granules (MRGs). Mitochondrial aminoacyl-tRNA synthetases (mito aaRSs) catalyze tRNA charging and are key components in mitochondrial gene expression. Mutations of mito aaRSs are associated with various human disorders. However, the suborganelle distribution, interaction network and regulatory mechanism of mito aaRSs remain largely unknown. Here, we found that all mito aaRSs partly colocalize with MRG, and this colocalization is likely facilitated by tRNA-binding capacity. A fraction of human mitochondrial AlaRS (hmtAlaRS) and hmtSerRS formed a direct complex via interaction between catalytic domains in vivo. Aminoacylation activities of both hmtAlaRS and hmtSerRS were fine-tuned upon complex formation in vitro. We further established a full spectrum of interaction networks via immunoprecipitation and mass spectrometry for all mito aaRSs and discovered interactions between hmtSerRS and hmtAsnRS, between hmtSerRS and hmtTyrRS and between hmtThrRS and hmtArgRS. The activity of hmtTyrRS was also influenced by the presence of hmtSerRS. Notably, hmtSerRS utilized the same catalytic domain in mediating several interactions. Altogether, our results systematically analyzed the suborganelle localization and interaction network of mito aaRSs and discovered several mito aaRS-containing complexes, deepening our understanding of the functional and regulatory mechanisms of mito aaRSs.

Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine.

Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution.

Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.

Numerous applications of noncanonical amino acids (ncAAs) in basic biology and therapeutic development require efficient protein biosynthesis using an expanded genetic code. However, achieving such incorporation at repurposed stop codons in cells is generally inefficient and limited by complex cellular processes that preserve the fidelity of protein synthesis. A more comprehensive understanding of the processes that contribute to ncAA incorporation would aid in the development of genomic engineering strategies for augmenting genetic code manipulation. In this work, we used a series of fluorescent reporters to screen a pooled Saccharomyces cerevisiae molecular barcoded yeast knockout (YKO) collection. Fluorescence-activated cell sorting enabled isolation of strains encoding single-gene deletions exhibiting improved ncAA incorporation efficiency in response to the amber (TAG) stop codon; 55 unique candidate deletions were identified. The deleted genes encoded for proteins that participate in diverse cellular processes, including many genes that have no known connection with protein translation. We then verified that two knockouts, yil014c-aΔ and alo1Δ, exhibited improved ncAA incorporation efficiency starting from independently acquired strains possessing the knockouts. Using additional orthogonal translation systems and ncAAs, we determined that yil014c-aΔ and alo1Δ enhance ncAA incorporation efficiency without loss of fidelity over a wide range of conditions. Our findings highlight opportunities for further modulating gene expression with genetic, genomic, and synthetic biology approaches to improve ncAA incorporation efficiency. In addition, these discoveries have the potential to enhance our fundamental understanding of protein translation. Ultimately, cells that efficiently biosynthesize ncAA-containing proteins will streamline the realization of applications utilizing expanded genetic codes ranging from basic biology to drug discovery.

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Genetic Code Expansion in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying, engineering, and modulating protein complexes and biosynthetic enzymes in this organism are largely underdeveloped. Genetic code expansion for the incorporation of unnatural amino acids (unAAs) into proteins can advance such efforts and, furthermore, enable additional controls of biological processes of the strain. In this work, we established the orthogonality of two widely used archaeal tRNA synthetase and tRNA pairs in KT2440. Following the optimization of decoding systems, four unAAs were incorporated into proteins in response to a UAG stop codon at 34.6-78% efficiency. In addition, we demonstrated the utility of genetic code expansion through the incorporation of a photocross-linking amino acid, p-benzoyl-l-phenylalanine (pBpa), into glutathione S-transferase (GstA) and a chemosensory response regulator (CheY) for protein-protein interaction studies in KT2440. This work reported the successful genetic code expansion in KT2440 for the first time. Given the diverse structure and functions of unAAs that have been added to protein syntheses using the archaeal systems, our research lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Activity-Induced Cortical Glutamatergic Neuron Nascent Proteins.

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function.SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms.