Education

Research Interests

Control of enzyme activity and gene expression; mechanisms of enzyme catalysis; antimicrobial agents

A major focus of our research is the mechanism of protein splicing. Protein splicing, which was discovered only in 1990, is an unusual process by which the flow of information from a gene to its protein product is modulated post-translationally so as to yield two functionally unrelated proteins. It involves the precise, self-catalyzed excision of an intervening polypeptide sequence, the intein, from an inactive precursor protein with the concomitant joining of the flanking sequences, the exteins, to produce a new functional protein (see the diagram below). All information and catalytic groups required for protein splicing reside in the intein and the two flanking amino acids. The excised intein often functions as homing endonuclease, a property which makes inteins infectious elements that can be transferred horizontally between organisms and even species.

Click on diagram for a larger image.

All information and catalytic groups required for protein splicing reside in the intein. In the period 1993-96, we succeeded in defining each of the steps in the protein splicing process by isolating and characterizing the reaction intermediates. Protein splicing is a complex four-step process, which involves (1) N-S or N-O rearrangement of a peptide bond adjacent to a Cys or Ser residue to yield a linear peptide ester, (2) transesterification with a Cys, Ser, or Thr residue at the downstream splice junction to yield a branched ester intermediate, (3) cyclization of an Asn residue coupled to peptide bond cleavage, and (4) rearrangement of the transient splicing products to yield stable polypeptides. The first three reactions are catalyzed by the intein, but the final product rearrangement is a spontaneous, thermodynamically favored reaction that assures the irreversibility of protein splicing. These four reaction steps are outlined below:

Click on diagram for a larger image.

Inteins can be viewed as a class of highly unusual enzymes: (1) they catalyze three mechanistically distinct reactions; (2) they act on amino acid residues at their own N- and C-termini, so that the intein enzymes are also their own substrate, analogous to the role of catalytic RNA in the self-splicing of group I introns; and (3) their catalytic center comprises the two extremities of a polypeptide chain, a situation which is rarely encountered in conventional enzymes and suggests an unusual protein structure. Our aim is to elucidate the mechanism of catalysis of protein splicing by defining the catalytic center of the self-splicing intein, both in terms of the amino acid side chains involved and their arrangement in 3-dimensional space.

The experimental system used in our studies is the intein from the RecA protein of Mycobacterium tuberculosis. As a first step in our investigation, we cloned the RecA intein between two affinity tags as artificial exteins and genetically dissected away the portions of the intein that are involved in its homing endonuclease function so as to generate a minimal protein splicing element. Further dissection of the protein splicing element into separate N- and C-terminal fragments (about 100 amino acids each) showed that protein splicing can also occur in trans. This allowed us to develop an efficient in vitro trans-splicing system in which purified N- and C-terminal intein fragments are reconstituted and allowed to undergo splicing (see the diagram below). Under appropriate conditions, reconstitution and protein splicing can be studied separately, thus opening the way for analyzing both the structural and the catalytic basis of protein splicing.

Click on diagram for a larger image.

One-third of the world's population is infected with Mycobacterium tuberculosis and 5-10% of those infected suffer active tuberculosis, with nearly 3 million deaths annually. An alarmingly growing number of patients in developed country are suffering from multidrug-resistant TB, which is refractory to the mainline anti-tuberculosis drugs. Two important proteins of M. tuberculosis, DnaB and RecA, which are essential for DNA replication and DNA repair, respectively, are interrupted by closely related inteins. The excision of these inteins by protein splicing is required for the function of these proteins and the inhibition of protein splicing will therefore be lethal to this parasite. We are developing screening systems for inhibitors of RecA and DnaB intein-mediated protein splicing. Such inhibitors would constitute a new class of anti-mycobacterial drugs which may provide an effective treatment of multi-drug resistant tuberculosis.

Selected Publications

Xu, M.Q., Comb, D.G., Paulus, H., Noren, C.J. , Shao, Y. and Perler, F.B. (1994) Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. EMBO J, 13, 5517-5522.

Shao, Y., Xu, M.Q. and Paulus, H. (1995) Protein splicing: Characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence. Biochemistry 34, 10844-10850.

Shao, Y., Xu, M.Q. and Paulus, H. (1996) Protein splicing: Evidence for an N-O acyl rearrangement as the initial step in the splicing process. Biochemistry 35, 3810-3815.

Kochhar, S. and Paulus, H. (1996) Lysine-induced premature transcription termination in the lysC operon of Bacillus subtilis. Microbiology 142, 1635-1639.

Chong, S., Shao, Y., Paulus, H., Benner, J., Perler, F.B. and Xu, M.Q. (1996) Protein splicing involving the Saccharomyces cerevisiae VMA intein: The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vivo splicing system. J Biol Chem 271, 22159-22168.

Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R., Hirvonen, C.A., Pelletier, J.J., Paulus, H. and Xu, M.Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271-281.

Shao, Y. and Paulus, H. (1997) Protein splicing: Estimation of the rate of O-N and S-N acyl rearrangements, the last step of the splicing process. J. Peptide Res 50, 193-198.

Perler, F., Xu, M.Q. and Paulus, H. (1997) Protein splicing and autoproteolysis mechanisms. Curr Opin Chem Biol 1, 292-299.

Shingledecker, K., Jiang, S.-q. and Paulus, H. (1998) Molecular dissection of the Mycobacterium tuberculosis RecA intein: Design of a minimal intein and of a trans-splicing system involving two intein fragments. Gene 207, 187-198.

Paulus, H. (1998) Protein splicing: A novel form of gene expression and paradigm for self-catalyzed protein rearrangements Pure Appl Chem70, 1-8.

Mills, K.V., Lew, B.M., Jiang, S.-q. and Paulus, H. (1998) Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein. Proc Natl Acad Sci USA 95, 3543-3548.

Paulus, H. (1998) The chemical basis of protein splicing. Chem. Soc. Rev. 27, 375-386.

Lew, B.M., Mills, K.V. and Paulus, H. (1998) Protein splicing in vitro with a semisynthetic two-component minimal intein. J Biol Chem 238, 15887-15890.

Guan, C., Liu, Y., Shao, Y., Cui, T., Liao, W., Ewel, A., Whitaker, R. and Paulus, H. (1998) Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase. J Biol Chem 273, 9695-9702.

Lew, B.M., Mills, K.V. and Paulus, H. (1999) Characteristics of protein splicing in trans mediated by a semisynthetic intein. Biopolymers (Peptide Science) 51, 355-362.

Shingledecker K., Jiang, S.-q. and Paulus, H. (2000). Reactivity of cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein. Arch Biochem Biophys 375, 138-144.

Paulus, H. (2000). Protein splicing and related forms of protein autoprocessing. Annu Rev Biochem 69, 447-495.

Xu, M.Q., Paulus, H. and Chong, S. (2000). Fusions of self splicing inteins for protein purification. Meth Enzymol 326, 376-418.

Sun, L., Ghosh, I., Paulus, H. and Xu, M.Q. (2001) Protein trans-splicing to produce herbicide-resistant acetolactate synthase. Appl Environ Microbiol 67, 1025-1029.

Mills, K. and Paulus, H. (2001) Reversible inhibition of protein splicing by zinc ion. J Biol Chem 276, 10832-10838.

Paulus, H. (2001) Inteins as enzymes. Bioorg Chem 29,119-129.

Gangopadhyay, J.P., Jiang, S.-q., van Berkel, P. and Paulus, H. (2003) In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without amino acid substitutions at the splice junctions. Biochim Biophys Acta 1619, 193-200.

Gangopadhyay, J.P., Jiang, S.-q. and Paulus, H. (2003) In vitro screening system for protein splicing inhibitors based on green fluorescent protein as indicator. Analytical Chem 75, 2456-2462.

Paulus, H. (2003) Inteins as targets for potential antimycobacterial drugs. Frontiers in Bioscience 8, s1157-1165.