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Signal Peptides 299 that binds denatured mature portions of proteins. 17,26,27 If so, how does SecB discriminate preproteins from others? To reconcile the paradox, a kinetic partitioning model, which indicates that retardation of protein folding by signal peptides is the major factor for SecB to discriminate its substrates, has been proposed by Hardy and Randall. 28,29 However, the binding rate of SecB is much faster than rates of protein synthesis and its folding 21 and the substrate specificity of SecB does not seem to explain its in vivo preference with preproteins. 27 Mutations on signal peptides or on early mature regions can change apparent SecB dependency; thus the ability to interact with the transport machinery of a protein may be the key factor to determining its apparent SecB dependency. 30 Recently, the crystal structure of SecB from Haemophilus influenzae was determined, 31 which shed some light on this paradox, although the structure binding its substrate would be desirable for detailed discussion. 18,19 SecB is a highly acidic homotetramer. A mutational study implies that the tetramer is a dimer of dimers, 32 which was confirmed by the x-ray structure. In its quaternary structure, SecB seems to have two long grooves that are proposed to be the unfolded polypeptide binding sites. 31 The groove consists roughly of two subsites: one lined with conserved aromatic residues, the other a shallow floor rich in hydrophobic residues. Possibly, proteins in their translocation-competent state wrap around SecB. The SecB tetramer also has two potential SecA-binding sites, which are solvent-exposed flat surfaces rich in acidic residues. In addition, four exposed arms of SecB may embrace the SecA protein. Thus, subsequent binding among SecB, SecA, and preprotein could be the determinant of specific signal recognition. 14.2.1.2 SecA In Escherichia coli, SecA is a large protein (901 residues) that works as a homodimer. 33,34 SecA is a part of the bacterial protein translocase complex; it binds to SecY through its N-terminal domain but also loosely binds to anionic phospholipids possibly through its C-terminal domain. 35-37 The exact role of SecA in preprotein translocation has not been fully understood; its RNA helicase activity is not required in vivo for efficient protein translocation. 38 SecA also has the ATPase activity: its binding to SecB 39 and to anionic phospholipids 40,41 induces its conformational change, thus stimulating its ATPase activity, especially in the presence of SecYEG. Synthetic signal peptides were originally reported as inhibitors of ATPase activity without their mature portion, 40,42 although some recent experiments show that they stimulate ATPase activity in the presence of lipids. 43,44 However, a soluble N-terminal fragment of SecA was shown to bind signal peptides more tightly and this binding inhibits its ATPase activity. 45 It has been regarded that SecA is an ATP-driven motor protein and pushes preproteins into the membrane. 46 According to the membrane insertion hypothesis, SecA cycles insertion and deinsertion processes with the preprotein by utilizing the driving force of ATP hydrolysis, 47,48 but the detailed mechanism has yet to be elucidated. 19 As another driving force, the proton motive force (pmf) also seems to contribute significantly to the (late stage of) translocation process. 49
300 Cell-Penetrating Peptides: Processes and Applications 14.2.1.3 SecYEG and Related Proteins Three integral membrane proteins, SecY (PrlA), SecE, and SecG constitute the SecYEG complex, which is the central component of the translocase (or translocon). 19,34,50 This complex plays an active role in protein translocation because some mutations of SecY can affect the topology of substrate membrane proteins. 51 Other proteins, including YidC 52 and the SecDFyajC (SecD, SecF, and YajC) heterotrimer, 53 are also involved. There remain possibilities of yet uncharacterized factors. SecYEG is likely to exist in both monomeric and tetrameric forms. 54 Controversy still exists on whether the SecYEG monomer constitutes a functional channel or works as a tetramer. 55,56 Homologous proteins of YidC exist in mitochondria (Oxa1p) and chloroplasts (Alb3) but not in ER. 52 Oxa1p and Alb3 play a key role in the assembly of mitochondrial inner membrane proteins and chloroplast thylakoid membrane proteins, respectively. YidC helps translocation of membrane proteins, possibly by clearing the channel of SecYEG translocase, thus preventing the jamming of its substrates; 57 however, it also seems to be able to translocate proteins independently from the translocase. The SecDFyajC complex controls SecA membrane cycling to regulate the movement of the translocating preprotein. 53 14.2.2 SRP-DEPENDENT PATHWAY IN BACTERIA Since it had been widely believed that protein translocation directed by signal peptides occurs post-translationally in prokaryotes, 10 the discovery of E. coli genes homologous to the SRP components lead to a great interest on whether they really play significant roles in protein translocation or not. With subsequent extensive analyses, it turned out that the bacterial SRP system is essential, selectively translocating a group of proteins. 58-60 An important point is that this system also recognizes signal peptides or the first transmembrane segments; thus, there must be some discrimination mechanism between signal peptides (see Section 14.4.3.1). 14.2.2.1 Ffh and 4.5S RNA Although potential involvement of additional proteins and RNAs cannot be ruled out, the bacterial SRP system (SRP and its receptor) basically consists of two proteins and one RNA. Like mammalian SRP, bacterial SRP is a ribonucleoprotein complex, but it simply consists of Ffh and 4.5S RNA. 61 Ffh is named as a homolog of the mammalian SRP54 subunit (i.e., Fifty-Four Homolog). It consists roughly of two major domains: the NG domain, which is related to GTPase activity and binding to FtsY, and the M domain, related to binding to 4.5S RNA and to signal peptides. A crystal structure of Ffh from a thermophilic bacterium, Thermus aquaticus, was determined. 61 Subsequently, a crystal structure of the complex between domain IV of 4.5S RNA and the M domain of Ffh was solved. 62 From previous studies on the amino acid sequence of Ffh, the M domain was proposed to contain the methionine-bristle, where the unbranched thioester side chain of methionine enables flexible accommodation of a variety of signal sequences like bristles of a brush. 4,63 From crystallographic studies, the speculation was partly confirmed; however, a probably
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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1 CONTENTS 0-8493-1141-1/02/$0.00+$
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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