<p>The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using <i>Pichia pastoris</i> was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the K_M^app is more than 10² higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.</p>

The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.

Staphylococcus aureus is a remarkably successful pathogen, accounting for an estimated 95,000 invasive infections annually in the U.S. alone. The burden of MRSA infections on public healthcare continues to rise, particularly with the continued spread of antibiotic resistant strains and the hyper-virulent CA-MRSA strains. The pathogenic nature of S. aureus can be attributed to the cache of virulence factors encoded within the genome of this organism. Typically, these are secreted toxins which directly interact with the host during infection, and facilitate pathogenesis. A previous screen in our laboratory investigating proteases in S. aureus identified a mutant in aminopeptidase Z as being attenuated in disease causation. Classically aminopeptidases function in the bioactivation/inactivation of proteins; and/or the utilization of imported peptides for cellular nutrition. We therefore hypothesize that cells deficient in one of these two processes would have decreased fitness levels, resulting in reduced virulence. We therefore sought to explore the role of PepZ in S. aureus; either in the processing of exogenous oligopeptides for nutrition, or in protein bioactivation/inactivation, and protein stability. We determined that S. aureus strains deficient in PepZ are less viable when cultured under conditions of starvation or while in competition for nutrients with the parent strain, and does not appear to be peptide driven. Using protein analysis approaches we have identified PepZ externalization, suggesting a potential for the aminopeptidase beyond the confines of the cell membrane. Additionally, we have also identified a potential role for PepZ in protein stability in this organism. Lastly, we present the essential role for PepZ in S. aureus virulence.