Jason W. Chin

Regulation of Cellular Metabolism by Protein Lysine Acetylation

Metabolic Regulation Through Acetylation Covalent modification of lysine residues in various proteins in the nucleus is a recognized mechanism for control of transcription. Now two papers suggest that acetylation may represent an important regulatory mechanism controlling the function of metabolic enzymes (see the Perspective by Norvell and McMahon). Zhao et al. (p. 1000) found that a large proportion of enzymes in various metabolic pathways were acetylated in human liver cells. Acetylation regulated various enzymes by distinct mechanisms, directly activating some, inhibiting one, and controlling the stability of another. Control of metabolism by acetylation appears to be evolutionarily conserved: Wang et al. (p. 1004) found that the ability of the bacterium Salmonella entericum to optimize growth on distinct carbon sources required differential acetylation of key metabolic enzymes, thus controlling flux through metabolic pathways. Regulation of enzymes by acetylation controls metabolic function in human liver cells. Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl–coenzyme A hydratase/3-hydroxyacyl–coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.

Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase–tRNACUA (MjTyrRS–tRNACUA) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase–tRNACUA (MbPylRS–tRNACUA) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase–tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.

Genetically encoding N|[epsi]|-acetyllysine in recombinant proteins

N(epsilon)-acetylation of lysine (1) is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins containing N(epsilon)-acetyllysine (2) at defined sites. Here we demonstrate the site-specific incorporation of N(epsilon)-acetyllysine in recombinant proteins produced in Escherichia coli via the evolution of an orthogonal N(epsilon)-acetyllysyl-tRNA synthetase/tRNA(CUA) pair. This strategy should find wide applications in defining the cellular role of this modification.

A Method for Genetically Installing Site-Specific Acetylation in Recombinant Histones Defines the Effects of H3 K56 Acetylation

Summary Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many “bioorthogonal” reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3–7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN–tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.

Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click Chemistry

We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNA(CUA) pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, containing a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, containing an azido group, into recombinant proteins in Escherichia coli. Proteins containing the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddition reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chemically orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topology and structure by intramolecular orthogonal conjugation reactions.

Bioorthogonal reactions for labeling proteins.

O the past 15 years a great deal of progress has been made on the discovery, rediscovery, and invention of bioorthogonal reactions between functional groups that do not react with biological entities under physiological conditions but selectively react with each other. Strategies for labeling different classes of biomolecules have been developed by coopting the biosynthetic machinery of cells to introduce molecules containing bioorthogonal functional groups. Tagging approaches have allowed some additional functional groups to be attached to proteins, and genetic code expansion and reprogramming have facilitated the site-specific incorporation of unnatural amino acids bearing bioorthogonal functional groups into proteins in bacteria, mammalian cells, and animals via the discovery and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs and orthogonal ribosomes. In addition, selective pressure incorporation and its derivatives have allowed the statistical labeling of proteins and proteomes with analogues of natural amino acids. The incorporation of unnatural amino acids bearing bioorthogonal functional groups and their chemoselective labeling has great potential for imaging and controlling individual proteins and labeling proteomes, but the ability of investigators to leverage these approaches for biological discovery will be crucially dependent on the properties of the chemical reactions used. The reactants in a bioorthogonal reaction should be kinetically, thermodynamically, and metabolically stable before the reaction takes place and not toxic to living systems. The reaction should yield stable covalent linkages with no or innocuous byproducts. Moreover, the two bioorthogonal moieties have to react selectively with each other under physiological conditions (ambient temperature and pressure, neutral pH, aqueous conditions), without either of them crossreacting with the plethora of chemical functionalities found in living cells. Despite the challenges of meeting these criteria, a number of reactions have been developed that show good biocompatibility and selectivity in living systems (see Figure 1). Some of these reactions are chemoselective with respect to many but not all biological functionalities and have been used to label proteins in vitro and on the cell surface, while other reactions have additionally been used for the more challenging task of labeling proteins inside cells or living animals. Most bioorthogonal reactions follow second-order kinetics, and their rates depend directly on the concentrations of both reaction partners as well as on the intrinsic second-order rate constant k2 [M −1 s−1] of the reaction. Rapid reactions with high second-order rate constants are therefore advantageous for labeling during biological processes that occur on a very short time scale or for the labeling of low abundance proteins. Lower abundance proteins can sometimes be labeled with a large excess of labeling reagent, but this strategy may be practically limited by solubility, off target reactions and toxicity. Bioorthogonal reactions for which one partner can be installed into proteins are summarized in Figure 1. Their second-order rate constants span 9 orders of magnitude with the fastest bioorthogonal labeling reactions reaching rates up to 10 M−1 s−1, which approaches the rate constants for many enzymatic labeling approaches. Here we briefly introduce the bioorthogonal chemistries used for labeling proteins and comment on their utility for protein labeling before providing a perspective on future directions. Amongst the first functionalities to be explored as bioorthogonal reporters were ketones and aldehydes. Under acidic conditions (pH 4−6) their carbonyl groups react with strong α-effect nucleophiles such as hydrazines and alkoxyamines. Ketone/aldehyde condensations show rather slow kinetics with second-order rate constants in the range of 10−4 to 10−3 M−1 s−1, necessitating high concentrations of labeling reagent in order to achieve good labeling, which might be problematic in terms of toxicity and background signal. In general ketone/aldehyde condensations are best suited for in vitro or cell-surface labeling, because the reaction requires an acidic pH, which is difficult to obtain inside most cellular compartments. Furthermore, inside living cells, α-effect nucleophiles may undergo side-reactions with carbonyl-bearing metabolites. A functionality that is essentially absent from biological systems and truly orthogonal in its reactivity to the majority of biological functionalities is the azide group. Azide-bearing unnatural amino acids have been incorporated into proteins and used in a variety of chemical reactions. One potential limitation of the use of azides for protein labeling is that some unnatural amino acids bearing azides appear to be reduced in some proteins examined. Azide-modified proteins have been reacted with phosphines in Staudinger ligations. This reaction has been used to label biomolecules in living cells and animals. The Staudinger ligation, however, has slow kinetics: the reaction proceeds with second-order rate constants in the low 10−3 M−1 s−1 range. In addition many of the phosphine reagents are oxidized by air or metabolic enzymes. Azides can also react with terminal alkynes in [3 + 2] cycloadditions, catalyzed by Cu salts. The CuAAC (Cucatalyzed alkyne−azide cycloaddition) reaction proceeds considerably faster than the Staudinger ligation in physiological settings. However, its reliance on the Cu catalyst is not without problems, since Cu may be toxic to living systems, and decreasing the copper concentration is generally accompanied by a large decrease in reaction rate. The development of tailored water-soluble Cu ligands and/or

Expanding and Reprogramming the Genetic Code of Cells and Animals

Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

Substrate recognition by the AAA+ chaperone ClpB

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.