LEGISLACION NACIONAL

Because many diseases, including inflammationary diseases, arteriosclerosis, cancers and neurodegenerative diseases, have long been associated with altered proteolytic activity of proteases or disruption in the balance and control between protease activation and inhibition, it is highly-desirable to track protease signaling, protease activation and inhibition using very sensitive and effective methods. Dynamic visualization of intracellular protease activity can provide valuable information about their physiological roles and pathological processes. Objective and quantitative noninvasive imaging of protease activity would represent a significant

Figure 1. 1. Specificity diagram between enzyme and substrate.

The amino acid residues (Pn-Pn’) around the scissile bond in the substrate should have high complementary with the subsites of the enzyme.

advance towards rapid and dynamic screening as well as monitoring the effects of therapeutic agents. The studies of protease actions, signaling and pathways in living systems are frequently hindered by the lack of tools or probes capable of monitoring dynamic protease processes in various cellular locations.

Currently, protease activity is determined by several methods in cells. Real-time polymerase chain reaction (RT-PCR) (Gelmini, Tricarico et al. 2003; Peluffo, Young et al. 2005; House, Catchpole et al. 2007) and Western blot (Kossakowska, Edwards et al. 1998; Persad, Liu et al. 2004) were used to monitor the elevated gene expression, and protein expression of proteases and protease inhibitors, respectively. Although RT-PCR and Western blot are sensitive to assess the existence of proteases and their substrate products, limited information for understanding the dynamic activity of proteases in cells or in vivo is still provided.

One of the most commonly-used and commercially-available substrates for protease activity measurement is the application of chromogenic or fluorogenic peptide kits in cell lysate assays (Goddard and Reymond 2004). These methods generally measure the actions of most proteases in cell lysates using: chromophores conjugated to short peptides; peptide mimics encompassing enzymatic cleavage sites (Talanian, Quinlan et al. 1997; Thornberry, Rano et al. 1997). The most common chromophores linked to short peptide fragments of 3-6 amino acids that mimic the sequence encompassing the P1 to Pn cleavage sites (usually up to P3) are: p-

nitroaniline (pNA), 7-amino-4-methylcoumarin (AMC), 7-amino-4-trifluromethyl coumarin (AFC), rhodamine or fluorescein isothiocyanate-labeled casein (FITC). Although newly- developed peptide probes are able to penetrate cells, these probes are not ideal for the continuous dynamic imaging of enzyme actions due to limited lifetime, specificity and stability of these protease substrates, resulting from a lack of defined structure in solution because of their short

sequences. However, these short peptides usually degrade rapidly and can not be delivered into specific sub-cellular locations. Moreover, determination of protease activity using the chromophores conjugated with the mimic, small peptide dye is accomplished through the elimination of quenching to emit the absorbance or fluorescence following protease cleavage of the linker. This method is also hampered by poor specificity due to the difficulties in considering the P’ region amino acid residues of the cleavage linker. On the other hand, the diagnosis of diseases, such as chronic pancreatitis, are largely restricted to later stages of the illness due to limitations inherent in the currently available peptide kits designed for the detection of trypsin activity in cell lysate (DiMagno 1988; Lemaitre and D'Armiento 2006). Activation of trypsinogen and chymotrypsinogen by caerulein was previously reported in pancreatic cancer cells, MIA PaCa-2 via PAR1 and the PAR pathway using cell lysate assays (Yamaguchi, Kimura

et al. 1989; Halangk, Sturzebecher et al. 1997; Kruger, Lerch et al. 1998; Namkung, Han et al. 2004; Yamasaki, Takeyama et al. 2006).

Due to the limitations in real-time determination of protease activation and inhibition, a powerful method using fluorescence resonance energy transfer (FRET) of fluorescent protein pairs has been extensively investigated to track protease activity in vitro and in vivo. FRET- based protease probes are created using a GFP pair connected by a enzymatic cleavage linker (Figure 1.2). The properties of fluorescent proteins that allow for cofactor-independent chromophore formation and expression to provide the capability of monitoring numerous cellular events in living cells or organisms via living cell imaging (Shimomura, Johnson et al. 1962; Chalfie, Tu et al. 1994; Inouye and Tsuji 1994; Wang and Hazelrigg 1994; Chalfie 1995; Tsien 1998; Akemann, Raj et al. 2001; Shimomura 2005). Taking advantage of wild type GFP’s resistance to the cleavage for proteases and denaturation, the cleavage of the peptide bond at the

Figure 1. 2. The model of FRET-based protease sensor.

FRET-based protease sensors are mostly constructed by fluorescent protein pair (eg. cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)) through the connection of a cleavage linker for proteases. Once the cleavage linker is cleaved, the change of fluorescence resonance energy transfer is used to monitor protease activity.

connecting linker between GFP pair results in a change of fluorescence resonance energy transfer. This method has been applied to track the activity of many proteases including trypsin (Pollok and Heim 1999; Eggeling, Jager et al. 2005), caspase-3 (Xu, Gerard et al. 1998; Harpur, Wouters et al. 2001; Luo, Yu et al. 2001), caspase-8 (Luo, Yu et al. 2003) and thrombin (Zhang 2004; Wang, Cao et al. 2005), and offers the advantage of being able to monitor the dynamic processes of protease activation in vitro and targeting of specific locations in vivo. Although FRET-based protease sensors can be used to track the protease activity in living organisms, this strategy has still not been extended to practical applications, and no FRET-based protease sensors are commercially-available. Additionally, the catalytic kinetic parameters (kcat, Km and

kcat/Km) of FRET-based protease sensors have not been extensively discussed in the literature.