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.
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.
The experimental system used in our current 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. Our discovery that protein splicing can occur in trans, so that split inteins can serve as a protein ligase, offers many interesting possibilities for protein engineering.
Antimycobacterial drug discovery
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. Three important proteins of M. tuberculosis , DnaB, SufB, and RecA, are interrupted by closely related inteins and the excision of these inteins by protein splicing is required for the function of these proteins. DnaB and SufB are essential for mycobacterial survival and RecA is essential for error-prone DNA repair, which is responsible for most mutations to drug-resistance. Accordingly, the inhibition of protein splicing is unlikely to be overcome by the development of drug resistance because this would require two simultaneous mutation in a low-mutation background. We have developed an assay system for inhibitors of protein splicing, based on the reconstitution of functional green fluorescent protein. This assay has been adapted as a high throughput screen, which is currently being used under the NIH Roadmap program to screen large chemical libraries for potential antimycobacterial drugs. These would constitute a novel class of anti-TB drugs that, unlike all other TB drugs in use, would not elicit drug-resitance.
Hedgehog protein autoprocessing
Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal, but has recently been found also to be involved in certain stem-cell cancers, including pancreatic, small cell lung, intestinal, and prostate, all of which are notoriously difficult to treat. One of the first steps in Hh signaling is the
autoprocessing of Hh protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent autocleavage reaction that leads to the production of the cholesterol ester of the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule. The first of these reactions is very similar to the first step in protein splicing; indeed, the Hh autoprocessing domain bears a close evolutionary and sructural relationship to inteins. Our aim is to identify compounds that attenuate Hh autoprocessing as a research tool for dissecting the role of autoprocessing and the attendant cholesterol modification in Hh function, both in the many different contexts of embryonic development and in Hh ligand dependent stem cell cancers. We have developed an in vitro homogeneous assay system which measures changes in fluorescence polarization that accompany the cholesterol-depended autocleavage of Hh protein and are using it as a high-throughput screen for identifying autoprocessing inhibitors. Of special interest will be the study of the effect of the inhibitors identified by this research on the growth of Hh-dependent endodermal tumor cell lines. If growth inhibition of the tumor cell lines should indeed be observed, this would suggest that Hh autoprocessing, as the first step in the Hh signaling pathway, could be a possible therapeutic target.
Paulus, H. (2000). Protein splicing and related forms of protein autoprocessing. Annu Rev Biochem 69, 447-495.
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.
Mills, K.V. and Paulus, H. (2005) Biochemical mechanisms of intein-mediated protein splicing, in "Homing Endonucleases and Inteins" (M. Belfort, V. Derbyshire, B. Stodddard and D. Wood, eds), Springer Verlag, pp. 233-255.
Paulus, H. (2007) Protein splicing inhibitors as a new class of antimycobacterial agents, Drugs of the Future, 32, 973-984.
Jiang, S.-q. and Paulus, H. (2010) A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of hedgehog protein autoprocecessing. J. Biomol. Screening, in press.