Membrane Translocation Domain

Membrane translocation domains have been identified in toxins and viruses and derived from signal sequences of secreted proteins. When derived from a signal seqnence the translocation domain contains hydrophobic sequences [146-148] while the toxin and viral translocation domains contain mostly basic residues [149,150]. 

In terms of targeting, membrane translocation domains lack specificity for particnlar cells or tissues. Therefore, these domains shonld be combined with targeting domains snch as those discussed in the previous section. In snch a constrnct, the targeting domain wiU ensnre a rapid accumulation at the surface of a specific cell type and the translocation domain will facilitate entry into the cytosol of the target cells. 

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BoNTs (150 kDa) consist of two polypeptide chains the heavy chain (HC, 100 kDa) and the light chain (LC, 50 kDa), linked with disulfide and non-covalent bonds. The amine end of the LC is responsible for intraneural enzymatic activity. The HC contains a membrane translocation domain (a 50 kDa amino-terminal polypeptide) and a receptor-binding part (a 50 kDa carboxy-terminal polypeptide) (DasGupta, 1990 Krieglstein et ah, 1994). BoNT/A forms dimers, trimers, and bigger structures. BoNT/E generally has a monomer structure, but sometimes forms dimers. BoNT/B is a dimer (Ledoux et ah, 1994). 

The HSV-1 virion is approximately 20 run in diameter and consists of four components envelope, tegument, capsid, and viral genome. The envelope is derived from the cellular membrane and contains approximately 12 viral glycoproteins essential for viral entry. The tegument is the protein layer between the capsid and the envelope and contains at least 10 viral proteins, including VP 16 (essential for transactivation and virion envelopment), VP22 (membrane translocation domain), and virion host shut off (vhs) protein. The capsid consists of 7 viral proteins and contains the linear dsDNA genome, which is 152 kb in size. 

Fig. 7. Two possible interpretations of the transitions of the translocator domain detected by the kinetics of binding to ISO membranes at 4°C. (A) Translocation. The states not in contact with the external medium are the states in contact with the internal medium. (B) Occlusion. The binding sites can be in a state where they are not accessible from either side of the membrane. The spots represent the substrate molecule, cyt and per represent the cytoplasmic and periplasmic side of the enzyme, respectively.

Figure 1 The mode of action for bacterial AB-type exotoxins. AB-toxins are enzymes that modify specific substrate molecules in the cytosol of eukaryotic cells. Besides the enzyme domain (A-domain), AB-toxins have a binding/translocation domain (B-domain) that specifically interacts with a cell-surface receptor and facilitates internalization of the toxin into cellular transport vesicles, such as endosomes. In many cases, the B-domain mediates translocation of the A-domain into the cytosol by pore formation in cellular membranes. By following receptor-mediated endocytosis, AB-type toxins exploit normal vesicle traffic pathways into cells. One type of toxin escapes from early acidified endosomes (EE) into the cytosol, thus they are referred to as short-trip-toxins . In contrast, the long-trip-toxins take a retrograde route from early endosomes (EE) through late endosomes (LE), trans-Golgi network (TGN), and Golgi apparatus into the endoplasmic reticulum (ER) from where the A-domains translocate into the cytosol to modify specific substrates.

Touhara, K. Binding of multiple ligands to pleckstrin homology domain regulates membrane translocation and enzyme activity of -adrenergic receptor kinase. FEBS Lett., 417, 243-248 (1997)... 

The N-terminal portion of the 522-residue polypeptide chain of colicin El appears to be required for transport into the membrane and the central part for binding to the receptor the channelforming property is characteristic of the C-terminal region.k A similar organization has been established for the smaller colicin N translocation domain, (residues 1-66), receptor domain, (residues 67-182), and pore-forming domain (residues 183-387). 

The structural organization of CNTs is functionally related to the fact that they intoxicate neurons via a four-step mechanism consisting of (1) binding, (2) internalization, (3) membrane translocation, and (4) enzymatic target modification (Figure 4) (Montecucco et al. 1994 Montecucco and Schiavo 1995 Rossetto et al. 2006). The LC is responsible for the intracellular catalytic activity, the amino-terminal 50kDa domain of the H chain (Hn) is implicated in membrane translocation, and the carboxy-terminal part (He) is mainly responsible for the neurospecific binding. 

Harris JB, Grubb BD, Maltin CA, Dixon R (2000) The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin. Exp Neurol 161 517-26 Haug G, Wilde C, Leemhuis J, Meyer DK, Aktories K et al. (2003) Cellular uptake of Clostridium botulinum C2 toxin membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain. Biochemistry 42 15284-91 Hauschild A (1993) Epidemiology of human foodborne botulism. In Hauschild A, Dodds KL (eds) Clostridium botulinum ecology and control in foods. Marcel Dekker, Inc. New York, pp 69-104... 

A1 and concanamycin A—two established V-ATPase inhibitors.163 This observation was confirmed by both in vitro enzyme assays and in vivo activity studies in mutant yeast. These studies indicated that, while the benzolactone enamides were potent V-ATPase inhibitors, a profound selectivity occurred between the mammalian and fungal enzymes. Subsequent studies by De Brabander determined that salicylihalamide A binds irreversibly to the trans-membranous proton-translocating domain via A-acyliminium chemistry.164... 

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