Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structureguided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.fluorescence microscopy | biotechnology | enzyme engineering T echniques for posttranslational labeling of proteins in living cells address some of the shortcomings of green fluorescent protein (GFP) by expanding the repertoire of chemical probes available for protein visualization (1). However, most of these methods, such as HaloTag (2), SNAP/CLIP (3), and DHFR (4), still use large protein tags that sterically interfere with protein trafficking and function, as GFP is known to do (5, 6). FlAsH (7) is the only peptide-based posttranslational labeling method that currently works inside living cells. At 12 amino acids, the FlAsH tag is much smaller than GFP, but poor labeling specificity (7-9), cellular toxicity, and undesired palmitoylation (7) and oxidation (8) of the Cys 4 recognition motif limit its utility.Here we introduce a method for protein labeling that utilizes a peptide tag while preserving high sequence specificity inside living cells. Our method, called PRIME (PRobe Incorporation Mediated by Enzymes), is based on a "fluorophore ligase" that is engineered from the Escherichia coli enzyme lipoic acid ligase (LplA). LplA's natural function is to ligate lipoic acid onto three E. coli proteins involved in oxidative metabolism (10). We previously used LplA for labeling of cell-surface proteins by demonstrating that the wild-type enzyme can ligate an azidoalkanoic acid instead of lipoic acid (11) (Fig. 1A, Middle). Ligated azide could then be chemoselectively derivatized using cyclooctynefluorophore conjugates.In this work, we wished to extend LplA-mediated labeling to intracellular proteins but recognized the challenges associated with our two-step labeling scheme. First, labeling sensitivity is limited by the kinetics of strain-promoted ½3 þ 2 cycloaddition, which has a rate constant of 4.3 × 10 −3 M −1 sec −1 (12). Second, for intracellular applications, two washout steps would be needed: first to remove excess azide, and second to remove excess fluorophore. We s...

Dysregulated pH is a common characteristic of cancer cells, as they have an increased intracellular pH ( pH i ) and a decreased extracellular pH ( pH e ) compared with normal cells. Recent work has expanded our knowledge of how dysregulated pH dynamics influences cancer cell behaviors, including proliferation, metastasis, metabolic adaptation and tumorigenesis. Emerging data suggest that the dysregulated pH of cancers enables these specific cell behaviors by altering the structure and function of selective pH-sensitive proteins, termed pH sensors. Recent findings also show that, by blocking pH i increases, cancer cell behaviors can be attenuated. This suggests ion transporter inhibition as an effective therapeutic approach, either singly or in combination with targeted therapies. In this Cell Science at a Glance article and accompanying poster, we highlight the interconnected roles of dysregulated pH dynamics in cancer initiation, progression and adaptation.

E. coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine sidechains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides (Nat. Biotechnol. 2007, 25, 1483-1487 and photocrosslinkers (Angew. Chem Int. Ed Engl. 2008, 47, 7018-7021) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA's utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA's natural protein substrates have a conserved β-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called "LplA Acceptor Peptide" (LAP). A ~10 7 library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a "LAP2" sequence with a k cat /K m of 0.99 μM −1 min −1 , >70-fold better than our previous rationally-designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483-1487, and only 8-fold worse than the k cat /K m values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.

The intracellular pH (pHi) of most cancers is constitutively higher than that of normal cells and enhances proliferation and cell survival. We found that increased pHi enabled the tumorigenic behaviors caused by somatic arginine-to-histidine mutations, which are frequent in cancer and confer pH sensing not seen with wild-type proteins. Experimentally raising the pHi increased the activity of R776H mutant epidermal growth factor receptor (EGFR-R776H), thereby increasing proliferation and causing transformation in fibroblasts. An Arg-to-Gly mutation did not confer these effects. Molecular dynamics simulations of EGFR suggested that decreased protonation of His776 at high pH causes conformational changes in the αC helix that may stabilize the active form of the kinase. An Arg-to-His, but not Arg-to-Lys, mutation in the transcription factor p53 (p53-R273H) decreased its transcriptional activity and attenuated the DNA damage response in fibroblasts and breast cancer cells with high pHi. Lowering pHi attenuated the tumorigenic effects of both EGFR-R776H and p53-R273H. Our data suggest that some somatic mutations may confer a fitness advantage to the higher pHi of cancer cells.

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: PRobe Incorporation Mediated by Enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase site-specifically attaches a picolyl azide derivative to a 13-amino acid recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing picolyl azide are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize picolyl azide, perform PRIME labeling, and achieve CuAAC derivatization of picolyl azide on live cells, fixed cells, and purified proteins. Reagent preparations, including synthesis of picolyl azide probes and expression of lipoic acid ligase, take 12 d, while the procedure to perform site-specific picolyl azide ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.

Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3'-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels ( approximately 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus.

Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.

The International Society of Cancer Metabolism (ISCaM) meeting on Cancer Metabolic Rewiring, held in Braga Portugal in October 2019, provided an outstanding forum for investigators to present current findings and views, and discuss ideas and future directions on fundamental biology as well as clinical translations. The first session on Cancer pH Dynamics was preceded by the opening keynote presentation from our group entitled Intracellular pH Regulation of Protein Dynamics: From Cancer to Stem Cell Behaviors. In this review we introduce a brief background on intracellular pH (pHi) dynamics, including how it is regulated as well as functional consequences, summarize key findings included in our presentation, and conclude with perspectives on how understanding the role of pHi dynamics in stem cells can be relevant for understanding how pHi dynamics enables cancer progression.