Charles G. Miller

Intracellular proteins are degraded in all known cells. The mechanisms by which particular proteins are recognized for degradation, the pathways by which degradation occurs, and the nature and regulation of the enzymes involved are not well understood in any organism. In my lab, we use the well developed genetic systems of enteric bacteria along with biochemical and molecular biological methods to study these problems.

The protein degradation pathway. By isolating mutations that affect each of the major peptidase activities of Salmonella typhimurium, we have identified the genes and enzymes that act in the final steps of the degradation pathway to produce free amino acids from peptide intermediates. Nearly all of these genes have been cloned and several have been sequenced. The peptidase product of several of the genes has been purified. Much remains to be done, however, to fully characterize the properties of these genes and enzymes. We would like to know, for example, how each of the peptidase genes is regulated. Work currently underway is aimed at identifying and characterizing the endoproteases that produce the intermediates that are the substrates for the peptidases. We are also attempting to devise methods for identifying the intermediates formed in vivo from a single degradable protein. This will allow us to define, more precisely, where in the pathway individual peptidases act.

N-terminal modification. In eubacteria, protein synthesis is initiated with N-formyl methionine. The formyl group is nearly always removed before a protein reaches its mature form. In some proteins, methionine remains as the N-terminus of the mature protein, while in others the methionine residue is also removed. The enzyme that removes N-terminal methionine from newly synthesized peptide chains is an aminopeptidase different from those that act in protein degradation. This enzyme, the product of the pepMgene, is specific for N-terminal methionine, it requires one of a distinct group of amino acids in the position following methionine, and it removes only the N-terminal methionine without further cleavage of the peptide. This peptidase is required for growth and mutations in pepM are lethal. We are interested in understanding the processes of N-terminal modification in more detail. We would like to understand, for example, what happens to protein synthesis in a cell in which the methionine amino-peptidase doesnít work, at precisely what point in the synthesis of a polypeptide does the methionine aminopeptidase act, and if the methionine aminopeptidase interacts with other proteins to carry out its function.

Anaerobic regulation of peptidase gene expression. pepT, the gene for one of the Salmonella typhimurium peptidases (peptidase T, an aminotripeptidase), is transcribed at much higher levels under anaerobic conditions than in aerobically growing cells. This gene is a member of a family of genes that depends on a positive transcriptional regulator (the product of the oxrAor fnr gene) for increased anaerobic transcription. We are analyzing the mechanism of this regulation and are especially interested in a class of mutations in rpoA, the gene for the a-subunit of RNA polymerase, that affect anaerobic expression of pepT. These mutations relatively specifically affect oxrA-dependent transcription and all produce changes in the C-terminal region of the alpha protein. We believe that these mutations define a region of the alpha subunit that is directly involved in interactions between RNA polymerase and a transcriptional activator.

• Conlin, C.A., N.J. Trun, T.J. Silhavy & C.G. Miller (1992). Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene. J. Bacteriol. 174:5881-5887.
• Conlin, C.A., E.R. Vimr & C.G. Miller (1992). Oligopeptidase A is required for normal phage P22 development. J. Bacteriol. 174:5869-5880.
• Miller, C.G., & C.A. Conlin (1994). Signal Peptide Hydrolases. In, G. von Heijne, ed., Signal Peptidases, R. G. Landes, Austin.
• Conlin C.A., & C.G. Miller (1995). Dipeptidyl Carboxypeptidase and Oligopeptidase A from Escherichia coli and Salmonella typhimurium. Meth. in Enzymol. 248:567-579.
• Miller, C.G. (1996). Protein Degradation and Proteolytic Modification. In, Neidhardt, et al. eds. The genetics and molecular biology of Escherichia coli and Salmonella typhimurium. American Society for Microbiology Press, Washington.
• Lombardo, M-J., A.A. Lee, T.M. Knox and C.G. Miller (1997). Regulation of the Salmonella typhimurium pepT gene by cyclic AMP receptor protein (CRP) and FNR acting at a hybrid CRP-FNR site. J. Bacteriol. 179:1909-1917.
• Carinato, Maria E, P. Collin-Osdoby, X. Yang, T.M. Knox, C.A. Conlin & C.G. Miller (1998). The apeE Gene of Salmonella typhimurium Encodes an Outer Membrane Esterase Not Present in Escherichia coli. J. Bacteriol. 180:3517-3521.
• Wei, Yan & C.G. Miller (1999). Characterization of a Group of Anaerobically Induced, fnr-Dependent Genes of Salmonella typhimurium. J. Bacteriol. 181:6092-6097.
• Conlin, C.A. & C.G. Miller (2000). opdA, a Salmonella enterica serovar Typhimurium gene encoding a protease, is part of an operon regulated by heat shock. J.Bacteriol. 182:518-521.
• Lassy, R.A. & C.G. Miller (2000). Peptidase E, a peptidase specific for N-terminal aspartic dipeptides, is a serine hydrolase. J.Bacteriol. 182:2536-2543.
• Mathew, Z., T.M. Knox & C.G. Miller (2000). Salmonella enterica Serovar Typhimurium Peptidase B is a leucyl aminopeptidase with specificity for acidic amino acids. J.Bacteriol. 182:3383-3393.
• Hakansson, K., D. Broder, A.H.-J. Wang & C.G. Miller (2000). Crystallization of peptidase T from Salmonella typhimurium. Acta Cryst. 56:924-926.