Cytoplasmic peptidases

FIG. 1 Digestion and absorption of proteins in the small intestine. (1) Brush-border peptidases, (2) brush-border amino acid transport systems, (3) brush-border peptide transport systems, (4) cytoplasmic peptidases, (5) basolateral amino acid transport systems, (6) basolateral peptide transport systems. 

Luminal and Membrane Metabolism of Peptides and Proteins. In meaningful studies on peptide and protein drug absorption in the small intestine, it is prerequisite to distinguish among cavital, membrane contact, and intracellular drug metabolism.Cavital metabolism takes place in the lumen of the small intestine by enzymes such as trypsin, chymotrypsin, carboxypepti-dase, and elastase, which are secreted by the pancreas. Membrane contact metabolism is carried out by aminopeptidases lo-calized on the brush border membrane. Intracellular metabolism occurs inside of the cells. The known intra-celluar enzymes are cytoplasmic peptidases, prolidase, dipeptidase, and tripeptidase.A more detailed dis-cussion of this topic is presented in section Intestinal Absorption Barriers, later. 

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

Ajami, K., Abbott, C.A., McCaughan, G.W. and Gorrell, M.D. (2004) Dipeptidyl peptidase 9 has two forms, a broad distribution, cytoplasmic localization and DPIV-like peptidase activity. Biochimica et Biophysica Acta, 1679, 18-28. 

The proteolytic activity of some multicatalytic peptidases is stimulated by ATP, whereas that of others is not influenced by ATP [32], The ATP-dependent proteolytic system first found in reticulocytes requires the presence of a heat-stable polypeptide called ubiquitin, one of the roles of which is to mark particular proteins for subsequent degradation [33. ATP-Indepen-dent multicatalytic peptidases can degrade proteins that have a free amino or an /V-acctylatcd terminus, as well as oxidatively altered or phosphorylat-ed proteins [34], The small peptides generated are resistant to multicatalytic peptidases and are further degraded by cytoplasmic endopeptidases and exopeptidases. 

During the last ten years, it has become apparent that calcium-dependent papain-like peptidases called calpains (EC 3.4.22.17) represent an important intracellular nonlysosomal enzyme system [35][36], These enzymes show limited proteolytic activity at neutral pH and are present in virtually every eukaryotic cell type. They have been found to function in specific proteolytic events that alter intracellular metabolism and structure, rather than in general turnover of intracellular proteins. Calpains are composed of two nonidentical subunits, each of which contains functional calcium-binding sites. Two types of calpains, i.e., /i-calpain and m-calpain (formerly calpain I and calpain II, respectively), have been identified that differ in their Ca2+ requirement for activation. The activity of calpains is regulated by intracellular Ca2+ levels. At elevated cytoplasmic calcium concentrations, the precursor procal-pain associates with the inner surface of the cell membrane. This interaction seems to trigger autoproteolysis of procalpain, and active calpain is released into the cytoplasm [37]. 

Mitochondria arise by division and growth of preexisting mitochondria. Because they synthesize only a few proteins and RNA molecules, they must import many proteins and other materials from the cytoplasm. A mitochondrion contains at least 100 proteins that are encoded by nuclear genes.50,50a The mechanisms by which proteins are taken up by mitochondria are complex and varied. Many of the newly synthesized proteins carry, at the N terminus, presequences that contain mitochondrial targeting signals51-53 (Chapter 10). These amino acid sequences often lead the protein to associate with receptor proteins on the outer mitochondrial membrane and subsequently to be taken up by the mitochondria. While the targeting sequences are usually at the N terminus of a polypeptide, they are quite often internal. The N-terminal sequences are usually removed by action of the mitochondrial processing peptidase (MPP) in... 

Penicillins have been considered for the inhibition of other bacterial serine enzymes than the DD-peptidases and /3-lactamases. For instance, bacterial signal peptidases (SPases) are essential for cell viability and therefore represent nowadays a class of novel antibacterial target <1998NAT186>. SPases are involved in protein translocation through the cytoplasmic membrane in the final step of the bacterial protein secretion pathway <1997PSC1119>. 5(S)-Stereoisomers of penems have been found to inhibit SPases <1995BML443>. The most potent inhibitors are 5/AV-tricyclic penems <2003S1732>. 

In . coli, the signal peptides of some envelope proteins are cleaved cotranslationally, while other precursors are completely synthesized before processing can be detected. While MBP displays both modes of processing, cotranslational processing does not occur until the precursor is 80% complete (Josefsson and Randall, 1981). Since the catalytic domain of signal peptidase is on the periplasmic face of the cytoplasmic membrane (Ohno-Iwashita and Wickner, 1983), cotranslational pro-... 

Cystatin refers to a diverse family of protein cysteine protease inhibitors. There are three general types of cystatins Type 1 (stefens), which are primarily found in the cytoplasm but can appear in extracellular fluids Type 2, which are secreted and found in most extracellular fluids and Type 3, which are multidomain protease inhibitors containing carbohydrates and that include the kininogens. Cystatin 3 is used to measure renal function in clinical chemistry. See Barrett, A.J., The cystatins a diverse superfamily of cysteine peptidase inhibitors, Biomed. Biochim. Acta 45,1363-1374,1986 Katunuma, N., Mechanisms and regulation of lysosomal proteolysis, Revis. Biol. Cellular 20, 35-61, 1989 Gauthier, F., Lalmanach, G., Moeau, T. et al., Cystatin mimicry by synthetic peptides, Biol Chem. Hoppe Seyler 373, 465-470, 1992 Bobek, L.A. and Levine,... 

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