Production of cyclic peptides and proteins in vivo

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Premotor Mechanisms for Orofacial Coordination

The mouth, throat, and face contain numerous muscles that participate in a large variety of orofacial behaviors. The jaw and tongue can move independently, and thus require a high degree of coordination among the muscles that move them to prevent self-injury. However, different orofacial behaviors require distinct patterns of coordination between these muscles. The method through which motor control circuitry might coordinate this activity has yet to be determined. Electrophysiological, immunohistochemical, and retrograde tracing studies have attempted to identify populations of premotor neurons which directly send information to orofacial motoneurons in an effort to identify sources of coordination. Yet these studies have not provided a complete picture of the population of neurons which monosynaptically connect to jaw and tongue motoneurons. Additionally, while many of these studies have suggested that premotor neurons projecting to multiple motor pools may play a role in coordination of orofacial muscles, no clear functional roles for these neurons in the coordination of natural orofacial movements has been identified.

In this dissertation, I took advantage of the recently developed monosynaptic rabies virus to trace the premotor circuits for the jaw-closing masseter muscle and tongue-protruding genioglossus muscle in the neonatal mouse, uncovering novel premotor inputs in the brainstem. Furthermore, these studies identified a set of neurons which form boutons onto motor neurons in multiple motor pools, providing a premotor substrate for orofacial coordination. I then combined a retrogradely traveling lentivirus with a split-intein mediated split-Cre recombinase system to isolate and manipulate a population of neurons which project to both left and right jaw-closing motor nuclei. I found that these bilaterally projecting neurons also innervate multiple other orofacial motor nuclei, premotor regions, and midbrain regions implicated in motor control. I anatomically and physiologically characterized these neurons and used optogenetic and chemicogenetic approaches to assess their role in natural jaw-closing behavior, specifically with reference to bilateral masseter muscle electromyogram (EMG) activity. These studies identified a population of bilaterally projecting neurons in the supratrigeminal nucleus as essential for maintenance of an appropriate level of masseter activation during natural chewing behavior in the freely moving mouse. Moreover, these studies uncovered two distinct roles of supratrigeminal bilaterally projecting neurons in bilaterally synchronized activation of masseter muscles, and active balancing of bilateral masseter muscle tone against an excitatory input. Together, these studies identify neurons which project to multiple motor nuclei as a mechanism by which the brain coordinates orofacial muscles during natural behavior.

Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells -- and only those cells -- to be labeled with azides. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affnity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins -- including proteins with previously unknown expression patterns -- expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.