Вы находитесь на странице: 1из 29

www.annualreviews.org

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

ANNUAL Furt he r REVIEWS Click here for quick links to Annual Reviews content online,
ANNUAL
Furt he r
REVIEWS
Click here for quick links to
Annual Reviews content online,
including:
• Other articles in this volume
• Top cited articles
• Top downloaded articles
• Our comprehensive search
personal use only.

Annu. Rev. Biochem. 2008. 77:701–26

First published online as a Review in Advance on February 27, 2008

The Annual Review of Biochemistry is online at biochem.annualreviews.org

This article’s doi:

10.1146/annurev.biochem.75.103004.142532

Copyright

c 2008 by Annual Reviews.

All rights reserved

0066-4154/08/0707-0701$20.00

CFTR Function and Prospects for Therapy

John R. Riordan

Department of Biochemistry and Biophysics, Cystic Fibrosis Treatment and Research Center, School of Medicine, University of North Carolina at Chapel Hill, North Carolina 27599; email: jack riordan@med.unc.edu

Key Words

ABC protein, anion channel, cystic fibrosis, ER quality control, molecular therapy, protein misfolding

Abstract

Mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel cause cys- tic fibrosis (CF). The multidomain integral membrane glycopro- tein, a member of the adenine nucleotide-binding cassette (ABC) transporter family, conserved in metazoan salt-transporting tissues, is required to control ion and fluid homeostasis on epithelial sur- faces. This review considers different therapeutic strategies that have arisen from knowledge of CFTR structure and function as well as its biosynthetic processing, intracellular trafficking, and turnover.

701

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Contents

INTRODUCTION

702

CYSTIC FIBROSIS

702

ABC TRANSPORTERS

702

CFTR STRUCTURE

703

CFTR FUNCTION

704

Primary Anion Channel Function .

705

Mechanism of Channel Regulation

706

Secondary Functions

707

BIOSYNTHETIC PROCESSING AND INTRACELLULAR TRAFFICKING

708

Synthesis, Conformational Maturation, and Endoplasmic Reticulum Export

709

Role of Molecular Chaperones

711

Traffic in Endocytic and Distal Secretory Pathways

713

PROSPECTS FOR THERAPY

715

CFTR Gene Replacement

715

Suppression of Premature Stop Mutations

716

Restoration of CFTR Folding and Function

716

Modulation of Other Epithelial Ion Transport

718

CONCLUSIONS AND OUTLOOK

718

INTRODUCTION

The purpose of this review is to summarize what has been learned over the past 20 years about the cystic fibrosis transmembrane con- ductance regulator (CFTR) and to consider how this knowledge is being applied to the de- velopment of new therapies for cystic fibrosis (CF). The basic ideas are to consider what the CFTR is, what it does, what is wrong with it in disease, and what might be done to fix it.

CFTR: cystic

fibrosis

transmembrane

conductance

regulator

F508: deletion of phenylalanine at position 508 of the CFTR amino acid sequence

ABC: adenine

nucleotide-binding

cassette

CYSTIC FIBROSIS

The clinical features and history of the dis-

ease since its recognition as a discrete en-

702

Riordan

tity in 1938 are extensively described else- where, including several recent, excellent

concise reviews (1–3). From our present per- spective, it is clear that virtually all indi- viduals diagnosed clinically with CF have mutations in both CFTR gene alleles. Al- though approximately 1500 different disease- associated mutations have been identified,

a single codon deletion, F508, is by far

the most common and is present on at least one allele in approximately 90% of the patients in some populations (http://www. genet.sickkids.on.ca/cftr). Thus, although there is a wide range in severity and manifes- tations of this disease that are dependent on other genetic and environmental influences, mutations in the CFTR gene are the funda- mental cause of the disease. The CFTR gly- coprotein, essential in the apical membrane of epithelial cells to maintain ion and fluid home- ostasis, is unique as it is the only member of the large adenine nucleotide-binding cassette (ABC) protein family known to function as an ion channel. The absence of this precisely reg-

ulated anion channel activity results in the fail- ure of ionic and water homeostasis on exocrine epithelial surfaces. This causes accumulations

of macromolecular secretions, which are de-

hydrated and in an altered physical state. This occurs in most exocrine tissues but with the most serious consequences in the pancreas, where failure of bicarbonate-rich fluid and en-

zyme secretion impair intestinal digestion and absorption, and in the airways of the lung. There, viscous mucus accumulations and col- onization by microorganisms cause damaging inflammatory responses and loss of function. The lack of a CFTR chloride channel in the sweat duct blocks salt reabsorption, making its elevated concentration in sweat diagnostic of the disease.

ABC TRANSPORTERS

Members of this very large and ubiquitous family of membrane transporters were first identified in entirely separate studies of bac- terial nutrient uptake (4) and of the resistance

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

of mammalian tumor cells to antineoplastic drugs (5, 6). Sequences of the DNA coding for subunits of the bacterial nutrient importers (7) indicated that they were constituted by soluble nucleotide-binding and membrane- integrated subunits (in addition to a periplas- mic nutrient-binding subunit). The sequence of the multidrug resistance P-glycoprotein, overexpressed in cancer drug-resistant cells, revealed the presence of domains similar to the nucleotide-binding and membrane- spanning subunits of the bacterial nutrient importers, the bacterial hemolysin toxin ex- porter (8), and Drosophila eye color genes se- quenced earlier. Other individual transport proteins sharing these sequence similarities were discovered, including those that con- tribute to drug resistance or are mutated in several human genetic diseases (9). The family name ABC was applied to reflect the presence in all members of two homologous nucleotide-binding domains (NBDs) (9). Genome sequencing has revealed the presence of thousands of these domains with 48 complete ABC proteins in the human genome (10). Although only a few have been characterized biochemically and in membrane transport assays, ABC transporters may be considered transport ATPases as they trans- port substances against a concentration gra- dient, and their ATPase activities are stim- ulated by the compound transported. Each transporter exhibits internal symmetry of pri- mary structure with one membrane-spanning domain (MSD) and one NBD in the N- and C-terminal halves. In many ABC trans- porters, both of the ATP-binding sites are hydrolytic, whereas in others, including the human ABCC subfamily to which the CFTR belongs, hydrolysis occurs at only one of the sites (11). Understanding of the fundamental duality in the structure of ABC transporters became much clearer when it was found that both of the two ATP-binding sites are composite sites contributed to by residues from each of the NBDs (12–15). Each complete nucleotide- binding site is formed at the interface between

the NBDs, with the binding altering their spa- tial relationship to each other as well as coin- ciding with molecular rearrangements within the domains (16–18). Hydrolysis and disso- ciation of products may then enable return to the initial unbound state. The nucleotide- induced changes in the NBDs may be coupled to conformational alterations in the MSDs, contributing to transport. A possible struc- tural basis of such coupling became evident from the atomic structures of several complete bacterial ABC transporters (19–22). These re- veal associations between three-dimensional (3D) structural elements in NBDs that change position on ATP binding and others (so- called coupling helices) in cytoplasmic loops (CLs) separating membrane-spanning he- lices. The propagation of these implied molecular motions in NBDs, through the coupling helices, to the membrane region may contribute to the transmembrane transport event.

CFTR STRUCTURE

High-resolution structures of eukaryotic ABC transporters have not been determined yet, primarily because of limitations in generating homogeneous monodisperse preparations of sufficient quality and quantity for large-scale crystallization trials. The CFTR is especially demanding in this regard because of its low natural abundance and low level of expression in heterologous expression systems in which it is fully active functionally. So far, sufficient amounts of the CFTR from mammalian cell expression systems have been purified to gen- erate two-dimensional crystalline arrays (23) as well as single particles analyzed by electron microscopy to provide low-resolution 3D structural information (Figure 1). The low- resolution structure obtained shows strong similarity to that of P-glycoprotein generated by similar methods (24). Two distinct crys- tal forms are observed, possibly reflecting dif- ferent nucleotide-bound states. The location of the R domain relative to the membrane- associated and nucleotide-binding regions

www.annualreviews.org CFTR Function and Prospects for Therapy

NBD: nucleotide-

binding domain

MSD: membrane-

spanning domain

CL: cytoplasmic

loop

703

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Bipartate

substructure 2 nm Density ( ) 4.0 3.5 3.0 2.5 Interface 2.0
substructure
2 nm
Density ( )
4.0
3.5
3.0
2.5
Interface
2.0

Figure 1

CFTR structure by cryoelectron microscopy, dimeric CFTR particles as purified from detergent, and the CFTR crystallized as a monomer (23). Slices through the 3D structure taken perpendicular to the C2 axis which is tilted slightly toward the viewer and to the left. The interface apparent between the two monomers in the structure is indicated by the dashed line. The bipartite substructure of the upper region is indicated by the double arrow in the second panel from the left. The gray surface of the structure is drawn at a density equivalent to 2σ; above the mean. Color coding of the internal structure revealed by the slices is as shown on the right. Figure provided by Robert C. Ford.

PKA: protein kinase

A

is indicated by the position of site-specific nano-gold labeling (25). Crystals are currently being analyzed by cryoelectron microscopy to obtain higher-resolution structures. A high- resolution structure has been obtained for the isolated nucleotide-binding domain 1 (NBD1) of the CFTR synthesized in bacteria (26). It has the same basic fold as that of the NBDs of many bacterial ABC proteins deter- mined earlier. Most significantly the Phe508 residue occupies a position on the surface of the wild-type domain, and its absence has only minor effects on the domain structure (27), as indicated in Figure 2. The R domain, which separates NBD1 and MSD2 in the primary structure, is highly un- structured and distinguished primarily by a conserved set of phosphorylation sites, which control the activation state of the channel (28). Whereas this 200-residue region is the least conserved part of the protein sequence, both the relative positions of the phosphory- lation sites (29) and the order-disorder pat- tern across the sequence are highly con- served and hence functionally significant (T. Hegedus, unpublished observations). Phosphorylation by protein kinase A (PKA)

reduces the already low α-helical content of

the domain (30, 31), and a highly informa-

704

Riordan

tive NMR study has recently shown that the α-helical propensities of stretches of residues adjacent to many of the individual phospho- rylation sites are reduced when these sites are phosphorylated (32). Importantly, there is a corresponding diminution in the interaction of several of these stretches with NBD1 on phosphorylation. However, only the R do- main and NBD1 were present in these exper- iments, and R domain phosphorylation may have structural impact involving other parts of the molecule as well. Although a 3D structure of the full-length CFTR, at sufficient resolu- tion to enable clear definition of the R domain, is required to further understand its mech- anism of action, computational studies of its structure can be informative (T. Hegedus, un- published observations).

CFTR FUNCTION

Despite its architectural similarity to other members of the large ABC transporter family, the CFTR is functionally distinct in perform- ing as an ion channel. Apparently conforma- tional movements within and between the two NBDs are coupled to rearrangements among membrane-spanning sequences, which, rather than changing affinities and sidedness of

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

allocrite-binding sites, shift the equilibrium between the actively gating (open) and quiet (closed) ion pore. Such a device would gate and conduct anions continually were it not for the unique R domain, which, until phos- phorylated by PKA, restrains channel gating. Thus, the probability of channel opening is controlled by the extent of R domain phos- phorylation at multiple sites, reflecting the balance between protein kinases and the phos- phatases acting on these sites. In the reabsorp- tive duct of the sweat gland, for example, PKA apparently normally dominates, resulting in a continually active channel favoring maximal salt recovery. In contrast, in secretory epithe- lia, the channel may be kept unphosphory- lated and inactive until stimuli elevating cyclic AMP make the action of PKA exceed that of the phosphatases.

Primary Anion Channel Function

When first identified at the primary structure level, it was stated that the CFTR might either be the chloride channel defective in CF or a regulator of a separate channel protein (33). Although there are many reports of influences of the CFTR on other channels and trans- porters (34, 35), strong evidence indicates that the CFTR polypeptide itself mediates a char- acteristic epithelial anion conductance. Prop- erties of its tightly regulated single-channel conductance have been thoroughly character- ized (36–38) and are not detailed here. It be- haves as an ohmic low-conductance channel characteristic of that observed in the apical membrane of epithelial cells of the tissues af- fected in the disease (39). This pathway for the movement of chloride ions is required in ei- ther the secretion or absorption of salt across these epithelia (40). The CFTR also plays an important role in HCO 3 secretion because it is permeant to the anion (41) and because it probably stimulates Cl /HCO 3 exchangers (42). The most obvious manifestation of the loss of this function is the impaired pancre- atic HCO 3 secretion in patients, but there is

Figure 2

F429 F409 F433 F508 R553 R555
F429
F409
F433
F508
R553
R555

G550

there is Figure 2 F429 F409 F433 F508 R553 R555 G550 Comparison of nucleotide-binding domain 1

Comparison of nucleotide-binding domain 1 (NBD1) structure of the wild-type and F508 CFTR. Structures for the wild-type (2BBO, red ) and F508 (1XMJ, purple) CFTR were aligned, and the indicated mutations aiding crystallization were reverted using PyMol (http://pymol. sourceforge.net/). The regulatory insertion (404–435), which is missing from the NBD1 crystal structure, is inserted from a loop database search (SYBL,Tripos Inc., CA). That loop (cyan) contains two α-helixes in good agreement with hydrophobic patch analysis of this region (189). The solubilizing and rescue mutations are numbered and represented by sticks (light and dark green). Phe508 is colored yellow. The major differences between the wild-type and mutant domains, exhibited by different conformations of the two loops, are indicated with thick arrows. Figure provided by Tamas Hegedus.

also reduced pH in the epithelial surface liq- uid of other tissues (43). The failure to alka- linize the fluid into which they are secreted may be an important factor preventing the normal processing of mucins and contribut- ing to their hyperviscosity (44). Although reg- ulation of ionic balance, hydration, and pH adjustment are believed to contribute, there

www.annualreviews.org CFTR Function and Prospects for Therapy

705

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

remains an urgent need to further elucidate the exact mechanisms whereby the wild-type CFTR enables the achievement of the normal physical state of mucins as they are secreted.

Mechanism of Channel Regulation

The CFTR is atypical both as an ion channel and as an ABC protein, having adopted the ba- sic ABC transporter structural architecture to generate a ligand-gated channel whose level of activity is quantitatively controlled by the phosphorylation state of its unique R domain. Phosphorylation by PKA has only a minor ef- fect on CFTR ATPase activity (45) and ap- parently does not act primarily by influencing binding or hydrolysis of the ATP ligand (11) but does promote the association of the two NBDs (46). However, this latter effect is not entirely responsible for channel activation by phosphorylation because that occurs with ac- tive C-terminally truncated constructs lacking NBD2 (47, 48). R domain phosphorylation al- ters its conformation as well as contacts with other parts of the protein (30–32, 49). The relationship between binding and hy- drolysis of the ATP ligand and channel gat- ing also has not yet been entirely elucidated. From a thermodynamic perspective, interpre- tations have been somewhat confounded from the outset by assumptions about the role of the ATPase activity of the protein. An anal- ogy with ABC transporters that couple ATP hydrolysis to the transport of substances (all- ocrites) and the observation that hydrolyzable nucleoside triphosphates were preferred lig- ands (50) led to suggestions that CFTR chan- nel opening was energetically coupled to ATP hydrolysis, a view that was elaborated by oth- ers (51). Observations of apparent nonequilib- rium in transitions between two open states of slightly different conductance were inter- preted to mean that the gating process was not in thermal equilibrium (52). This nonequi- librium in gating seemed consistent with the previous suggestion that gating was driven by ATP hydrolysis. However, the apparent sec- ond open conductance state could only be ob-

706

Riordan

served in strongly filtered records owing to

a fast flickering mode too rapid for detec-

tion of its fully open state, and kinetic anal- ysis with extended bandwidth provides no ev- idence of nonequilibrium in gating (53, 54). Indeed, there is strong evidence that CFTR single-channel gating is a reversible, ther- mally driven process (55) and that hydroly- sis is not required for gating (56). Gating and hydrolysis do not appear to coincide either ki-

netically or energetically. It is now generally agreed that hydrolysis is not involved in chan- nel opening (57, 58). However, studies focusing primarily on the role of the tightening and loosening of the association between the two NBDs

of the CFTR have retained the interpretation

that channel closing is dependent on the en- ergy liberated by the cleavage of the terminal phosphate from ATP (59). This view is based primarily on the assumption that gating of the CFTR is not a thermodynamic equilibrium process and the observation of a higher ac- tivation enthalpy for closing (70 kJ/mol) in Xenopus oocytes (60) than that (20 kJ/mol) determined earlier in planar lipid bilayers (55). It was suggested that the latter lower value may have reflected the measurement and analysis of brief flickering transitions within channel open bursts rather than burst closures (60), but this is not the case, because the clos- ing rates yielding the lower values agree very well with the burst durations determined by patch-clamp analyses in several other labo- ratories (61). Thus, the discrepancy between

the different E a values for channel closing re- mains to be explained. In any case, the esti- mated free energy input of 70 kJ/mol for channel closing does not correspond well with the free energy of ATP hydrolysis, and there

is no suggestion of the source of the additional

35 kJ/mol (60). Moreover, in addition to chan- nel closing occurring in the absence of ATP hydrolysis, there is also evidence that chan- nel closing is not obligatorily dependent on a

change in the association of the two NBDs. That is, channel opening and closing events occur in the complete absence of NBD2 (47).

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Thus, even though a model with direct one-to-one coupling between the formation and disruption of tight ATP-mediated inter- NBD association and channel opening and closing, respectively, fits well with general in- terpretations of ABC protein function (16, 62), it does not account for all of the cur- rent data. Initiation of channel opening does not require tightening of the interface be- tween NBDs, although this may prolong the open state. The closing of channels, which are maintained in the open state in this way, is facilitated by hydrolysis of the NBD2-bound ATP and probably also by the disengagement of the NBD1-bound ATP from the degener- ate signature sequence of NBD2. Similarly, the gating of the full-length channels in re- sponse to nonhydrolyzable ATP analogues as ligands (56) may also reflect dynamic events at the interface between the NBDs, but in neither case is the hydrolysis per se an inte- gral part of the gating mechanism. One reason that the precise relationship between the en- zymatic and channel activities of the CFTR are not yet more fully understood is that the enzymatic characterization of the protein is still very limited (11, 45, 63, 64), and there is uncertainty as to whether activity assayed with the purified, reconstituted protein accurately reflects that in its native environment. One very interesting proposal is that chan- nel activity may correlate more closely with adenylate kinase than ATPase activity of the protein (65). Apparent adenylate kinase ac- tivity has been assayed with isolated CFTR NBDs, but not with full-length protein (66, 67). Effects of nucleotide mixtures of adenylate kinase substrates and products on CFTR activity in patch-clamp exper- iments have been described (65). It has been suggested that channel gating would be thermodynamically more compatible with a dependence on adenylate kinase than ATPase activity (65). However, there is no ther- modynamic incompatibility in a so-called hydrolyzable-ligand-gated process in which hydrolysis of the ATP ligand is a simple switch enabling release of structural strain in the pro-

tein rather than an energy source (61). Never- theless, lending credence to the possible im- portance of adenylate kinase activity of other ABC proteins, as well as the CFTR, is the recent description of the presence and role of this activity in the Mre11/Rad 50 DNA repair complex (68).

Secondary Functions

In addition to its well-established ion chan- nel function, the CFTR has been proposed to have many other roles and either directly or indirectly impacts other cellular proteins and functions (34). Thus, the term pleiotropic has been applied to the CFTR, but it is uncer- tain if this is appropriate. There are certainly downstream effects in addition to altered an- ion permeation owing to CFTR function and dysfunction. However, it remains a challenge to identify at what level a given downstream alteration is connected to the CFTR protein itself or the anion conductance that it medi- ates. From the CF disease perspective, the in- fluence of the lack of the CFTR on conduc- tive epithelial Na + permeability mediated by ENaC channels has received a great deal of emphasis (69, 70). This is because the con- tinued or enhanced Na + absorption is pri- marily responsible for the dehydration of the airway surface, which impairs mucociliary clearance (71). The importance of this effect is emphasized by the fact that transgenic over- expression of an ENaC subunit in mouse lung mimics the dehydration and mucus plugging, which occurs in the CF lung. (72). In addition to serving as a chloride chan- nel itself, the CFTR may also regulate other chloride channels, but these have not been identified at the protein level (73). There is strong evidence that the CFTR has evolved as an epithelial ion channel and that it is most strongly expressed in the salt-transporting ep- ithelia of teleosts, elasmobranchs, amphibia, avia, and mammals. Nevertheless, in some assays of CFTR RNA or protein, its pres- ence has been detected in nonpolarized cells, including those of the immune system. For

www.annualreviews.org CFTR Function and Prospects for Therapy

707

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

Rev. Biochem.

Annu.

example, digestion of phagocytosed material by alveolar macrophages was reported to be defective in CF because of the absence of the CFTR from the membrane of the phago- some vacuole where it normally enables acid- ification by providing a counterion pathway (74). Detection of the CFTR in neutrophils and other hemopoietic cells has also been de- scribed (75). The possibility of a direct con- tribution of the CFTR to the function of immune or inflammatory cells is an impor- tant issue that needs to be clearly resolved be- cause of the apparent hyperinflammatory sta- tus of patients’ tissues prior to infection (76). However, it is still unclear whether this is due to inherent changes in the inflammatory cells themselves or a reaction to the altered state of epithelial surfaces owing to ionic and fluid changes and mucus accretion. The failure of the CF lung to clear infectious microorgan- isms is generally attributed to impaired mu- cociliary clearance, but in addition, the CFTR has been proposed to serve as a receptor for Pseudomonas aeruginosa (77). In this mecha- nism, binding of the microbe to the receptor is required for its internalization and stimula-

Wild type ΔF508 by University of Hawaii
Wild type
ΔF508
by University
of Hawaii

Figure 3

Localization of the wild-type and F508 CFTR in highly differentiated primary human epithelial cells. The wild-type CFTR is present and the F508 CFTR is absent from the apical surface of ciliated airway cells. Fully differentiated primary cultures were adenovirally transduced to express a tagged CFTR protein, and the CFTR was detected in isolated cells using a mouse antibody recognizing the tag, followed by goat antimouse Alexa Fluor 288 lgG conjugate. The immunofluorescence is shown in overlay with differential interference contrast and nuclear staining by TO-PRO 3 iodide. Figure provided by Martina Gentzsch.

708

Riordan

tion of the cytokine secretion response neces- sary for clearance (78). In addition to chloride and bicarbonate, believed to be the main physiological anions passing through the CFTR, prevention of the passage of other permeant species in CF also may contribute to the disease phenotype. An important example from the infectious disease perspective is the apparent failure of trans- port of the thiocyanate anion onto the surface of CF airway epithelium (79). This can re- sult in the lack of oxidative generation of an- tibacterial hypothiocyanite, which may nor- mally help eliminate Staphylococcus aureus and P. aeruginosa.

BIOSYNTHETIC PROCESSING AND INTRACELLULAR TRAFFICKING

When the CFTR gene was initially identified, the absence of the codon for Phe508 was rec- ognized on the sequencing of cDNA synthe- sized from the sweat gland RNA of a patient (33). This deletion was then detected in ap- proximately two thirds of CF chromosomes (80). Heterologous expression of full-length F508 CFTR cDNA indicated that, although the glycoprotein was synthesized, it acquired only core and not complex oligosaccharide chains and failed to be transported to the cell surface (81). These findings have been con- firmed and extended in many different mam- malian cell expression systems. The likelihood that the biosynthetic arrest of F508 oc- curred in vivo in patients’ tissues and was not just an artifact of heterologous overexpres- sion was supported by the observation that the protein did not reach the apical membrane of sweat duct cells in fresh skin biopsies (82). More recently this has been verified in intes- tine and airway from patients (83, 84). The differential localization of the wild-type and mutant protein is illustrated in Figure 3. Expression of F508 in Xenopus oocytes (85) and insect cells (86), which are main- tained at lower temperatures than mammalian cells, resulted in at least partial maturation

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

and some activity of the mutant protein at the plasma membrane. This suggestion that the mutation is temperature sensitive was confirmed by the observation of similar behavior when mammalian cells expressing the mutant protein were shifted to lower tem- perature (87). This partial rescue by temper- ature manipulation presaged similar effects of treatment of cells with osmolytes, such as glycerol and other so-called chemical chaper- ones (88). This, in turn, encouraged searches for small-molecule rescuing agents that may ultimately be effective in the treatment of patients (89). These efforts, which are on- going, provide hope for the development of drugs to overcome the misprocessing of mu- tant CFTRs.

Synthesis, Conformational Maturation, and Endoplasmic Reticulum Export

Extensive metabolic labeling and pulse-chase experiments on the CFTR heterologously ex- pressed in a variety of mammalian cells have revealed that 5 to 10 minutes are required for a complete core-glycosylated CFTR polypep- tide to be formed (90). Although unglyco- sylated chains can be synthesized in vitro, none are formed in cells, indicating that N- glycosylation of the two sites in EL4 occurs cotranslationally (81). Transformation to a form in which the high-mannose oligosaccha- ride chains have been trimmed and extended to large complex chains takes longer, with re- sultant higher-molecular-weight species first appearing only after nearly a half hour of chase time (91). Complete transformation requires at least two hours, and during this period, only approximately one third of the precursor nascent chains are converted to mature prod- uct (92). The remainder is ubiquitylated and degraded by the 26S proteasome (93, 94). In- deed, ubiquitylation begins before translation is complete (95), and it is now realized that the CFTR is scrutinized by complex quality control systems during its synthesis and as- sembly as well as throughout its lifetime in

the cell. These systems deal with most mem- brane and secretory proteins, but the CFTR seems to have particular difficulty in achieving

a state that satisfies all criteria for export from the endoplasmic reticulum (ER). Wild-type versions of other ABC transporters such as P- glycoprotein and MRP1, for example, mature and are exported with nearly 100% efficiency compared to the 33% for the CFTR in the same cells (96). Interestingly, however, dele- tion of the counterpart of Phe508 in each of these ABC proteins has the same effect as in the CFTR, which is completely blocked mat- uration and ER export (97). The most obvious feature distinguishing the CFTR from other ABC proteins is the R domain, which is largely unstructured (32), and, therefore, possibly may contribute to the diminished efficiency of CFTR maturation. However, simple deletion of large portions of the R domain does not improve this ef- ficiency and, in fact, reduces it further (98). Both intradomain folding and interdomain assembly, not unexpectedly, are required to achieve a state competent for ER export (47, 99) as recognized by COP II constituents, which coat vesicles that bud from ER exit sites. The vesicle coat protein sec24 recognizes a diacidic exit code (DAD) of three residues in the NBD1 of the CFTR, which when mu- tated prevents ER export (100). It is easy to imagine that F508 or other processing mu- tants might conformationally mask this exit signal or possibly others and thereby elicit the same effect as inactivating it directly by mutagenesis. However, there are likely addi- tional dimensions to the recognition mecha- nisms during the dynamic interplay between progression toward a native state and tagging for a degradative fate. The role of molecular chaperones in this balancing act is outlined in

a section below. When the nascent CFTR was first rec- ognized as a substrate for ubiquitylation and degradation by the proteosome (93), it was disappointing to observe that proteasome in- hibitors did not promote maturation and ER export. A similar result was recently reported

www.annualreviews.org CFTR Function and Prospects for Therapy

709

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

with the yeast Yor1p ABC protein into which the equivalent of the F508 mutation was in- troduced (101). Thus, inhibition of endoplas- mic reticulum-associated protein degradation (ERAD) generally does not promote ER ex- port, although there is one report that this may occur to a limited extent (102). Never- theless, to identify additional points of pos- sible intervention, it is necessary to elucidate the different components of the quality con- trol systems and the sequence in which they evaluate the state of the polypeptide and di- rect its elimination if it has not yet reached a mature state. The Cyr laboratory (103) has contributed substantially to the current un- derstanding of these events. Having earlier demonstrated the involvement of a ubiqui- tylation complex that interacts with Hsc70 in F508 degradation (104, 105), this group more recently characterized a second complex containing the E3 ubiquitin ligase, RMA1, the E2 ubiquitin conjugating enzyme, Ubc6e, and the membrane-associated Derlin-1 (99). Their data and those of a parallel study (106) indicate that the latter complex, which also associates with the AAA-ATPase P97 [the yeast Cdc48 counterpart (107)], recognized the nascent CFTR early in its synthesis. The first CFTR MSD appears to be the target, per- haps initially of Derlin-1 if integration with the second MSD has not yet occurred. Sig- nificantly, this targeting of the F508 CFTR occurs even when both MSDs have already been synthesized, implying that the mutation may disrupt their assembly as is also indicated by other studies (47). Although this impact of F508 is observed in truncation constructs in which NBD2 is not present (47), the mutation is also known to prevent compact folding of that domain in the full-length protein (108). Indeed, Younger et al. (99) postulate that this may be what is detected by the first quality control com- plex, which they characterized involving the E2 UbcH5, E3 CHIP, the chaperone Hsc70, and its cochaperone, Hdj2. In this scenario, there is first scrutiny of MSD1 by the Derlin- 1/RMA1 membrane-associated complex fol-

710

Riordan

lowed by recognition of incompletely folded NBD2 by the cytoplasmic Hsc70/CHIP com- plex. There may be additional contributors to the efficient ERAD of incompletely or aberrantly assembled nascent CFTRs. For ex- ample, as mentioned in the chaperone sec- tion below, although the lectin chaperone- based conformation-sensing system of the ER lumen is not a primary determinant of CFTR quality control, overexpression of the mannose-binding EDEM (ER degradation enhancing α-mannoside-like protein) does accelerate degradation (107). This pathway, which is known to direct the retrotransloca- tion, ubiquitylation, and proteolysis of other secretory glycoproteins (109), may help en- sure that little unprocessed CFTR accumu- lates in the ER. Because of its dominant contribution to disease, most attention has been focused on the impact of F508 on CFTR folding and assembly. Replacement of the Phe normally present at this position by each of all other amino acids, rather than simple deletion, revealed that the presence of many differ- ent residues were compatible with substan- tial folding of the isolated NBD1 domain and the full-length protein (108, 110). The pres- ence of the amino acid backbone regardless of the side chain, with the exception of that of tryptophan, enabled refolding of bacteri- ally expressed NBD1. The requirements for maturation of a full-length CFTR were some- what more stringent with charged residues and large hydrophobics other than Phe, al- lowing very little formation of mature protein. These observations imply that the side chain may contribute to an interdomain interaction. Although the variants that matured showed some level of channel activity (108), the Phe aromatic side chain apparently plays a specific role in CFTR channel gating kinetics (111). In light of earlier evidence that the overall conformation of the F508 CFTR is grossly altered as reflected by susceptibility to lim- ited proteolysis (112), the finding that the 3D structure of the F508 NBD was little altered was somewhat surprising (27). However, this

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

was also found to be the case with the isolated domains containing either F508S or F508R

substitutions (110). In both of these variants, only a local surface perturbation around the 508 position was evident, invoking the idea that the main impact of the mutation may be

in disrupting a crucial interaction between this

small patch on the surface of NBD1 and an- other part of the molecule. Consistent with this, Du et al. (108) found that F508 caused little change in the pro- tease sensitivity of NBD1 but instead caused NBD2 to become much more sensitive to di- gestion. This could reflect impairment of a di- rect NBD1/NBD2 interaction or an indirect effect of disruption of an NBD1 association elsewhere in the molecule. F508 has been found to increase the protease sensitivity of

MSD1, to which it is linearly adjacent, as well as of NBD2 (47). However, possible sites of specific interaction of the Phe508 containing

a surface patch of NBD1 have not yet been

identified. Because F508 has essentially the same effect on a maturation-competent C- terminally truncated form of the CFTR from which NBD2 is entirely absent as on the full- length protein (47), it seems that such sites are likely within the membrane-spanning do- mains. Sequences in the CLs of both MSD1 and MSD2 may interact with NBD1 (20). Of the several hundred disease-associated mis- sense mutants in the CFTR, many causing misfolding and missassembly, several occur in CLs, with an especially high incidence in CL4 (113). Some of these CL4 substitutions could preclude an important interaction between this loop and NBD1 as suggested schemat- ically in Figure 4. Thus, many process- ing mutations may disrupt the crucial struc- tural interface between the cytoplasmic and membrane-integrated domains of the CFTR.

Role of Molecular Chaperones

As the nascent CFTR polypeptide is synthe- sized, it encounters molecular chaperones on both sides of the ER membrane. Initially, in- teractions of the immature form of both the

Wild type MSD1 MSD1 NBD1 R
Wild type
MSD1
MSD1
NBD1 R

ΔF508

form of both the Wild type MSD1 MSD1 NBD1 R Δ F508 MSD2 NBD2 Figure 4
MSD2 NBD2
MSD2
NBD2
of both the Wild type MSD1 MSD1 NBD1 R Δ F508 MSD2 NBD2 Figure 4 Illustration

Figure 4

Wild type MSD1 MSD1 NBD1 R Δ F508 MSD2 NBD2 Figure 4 Illustration of wild-type and

Illustration of wild-type and F508 CFTR domain assembly. Requirements for specific interactions between NBD1 and MSD2 and NBD2 and MSD1 are emphasized. The Phe508 deletion precludes the first of these interactions, which in turn prevents the normal integration of the MSDs, and NBD2 is unable to associate with MSD1. Adapted and revised from Reference 47. Figure provided by Lisa Brown.

wild-type and F508 CFTR, with the major cytoplasmic chaperone Hsp70 (114), and the lumen-facing chaperone of the ER membrane calnexin (115) were detected. Not unexpect- edly because most of the CFTR mass is consti- tuted by the large cytoplasmic domains, which must be folded and assembled correctly, mul- tiple cytoplasmic chaperones and cochaper- ones have since been implicated in these pro- cesses (116). An Hsp70 cochaperone of the

www.annualreviews.org CFTR Function and Prospects for Therapy

711

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Hsp40 class, Hdj-2, was shown to diminish aggregation and promote an early stage of assembly, possibly by recruiting Hsc70 to the cytoplasmic surface of the ER mem- brane (105). The nucleotide exchange factor HspBP1 aids Hsp70-supported CFTR fold- ing (117). In addition to the positive role of Hsc70 and its cochaperones in maturation of CFTR cytoplasmic domains, they also com- plex with an E2 ubiquitin-conjugating en- zyme Ubc H5 and the E3 ubiquitin ligase CHIP to direct ubiquitylation, which targets the nascent CFTR for proteasomal degra- dation (104). Although the nascent F508 CFTR has a somewhat greater tendency than the wild type to participate in these Hsc70- based complexes, neither simple up- or down- regulation of the chaperone significantly pro- motes maturation of the mutant (118). A drug with some ability to counter Hsp70 inter- actions also had minimal if any effect on F508 maturation (119). The fact that a large proportion of wild-type nascent chains undergo a similar set of interactions and a degradative fate probably means that very fine-tuned manipulation of many of these steps would be required to shift the folding yield of the mutant to a level nearer the wild type. Significantly, progress toward that end has recently been made by manipulations of the other major cytoplasmic chaperone complex, downstream from Hsc70, the Hsp90 sys- tem (116). Indeed, earlier work showed that the Hsp90 inhibitor geldanamycin completely prevented the maturation of both the wild- type and F508 CFTR, resulting in the rapid proteasomal degradation of immature nascent chains (96). A detailed proteomic analysis of CFTR-binding partners revealed a predomi- nance of known members of the Hsp90 chap- erone complex, especially in association with F508 (116). Several of these members were up- and downregulated to test whether more subtle modulation of this network might pro- mote CFTR maturation or stability, which is completely disrupted by inhibition of Hsp90

712

Riordan

function with an ansamycin drug (96). RNAi knockdown of the p23 cochaperone, believed to be involved in Hsp90 client loading, fur- ther destabilized the mutant nascent chains, whereas overexpression had a modest stabi- lizing influence without any promotion of maturation. FKBP8, a member of the im- munophilin family, involved in folding of other Hsp90 clients, further reduced the amount of the immature CFTR. Shifting the level of the Ahal cochaperone, which stimulates Hsp90 ATPase activity, in

contrast to p23, which inhibits activity, had

a more pronounced impact on levels of both

immature and apparently mature F508 pro- teins. Whereas Ahal overexpression dimin- ished the amounts of both forms, reduction of the cochaperone level with RNAi increased both, resulting in some of the functional CFTR at the cell surface. This provides proof

of principle that modulation of the nascent CFTR chaperone environment can provide a degree of rescue of the F508 CFTR. It is in- teresting that this has been possible with the Hsp90 network, which is dedicated to altering conformations of proteins that have already reached a basal folded state. Thus, the CFTR

is a member of the somewhat selective Hsp90

clientele. Significantly, related ABC proteins, including P-glycoprotein and multidrug resis- tance protein 1, are not dependent on Hsp90 for their maturation and stability (96). Thus, there is an apparent correlation between the inefficient maturation of the CFTR and its dependence on Hsp90. However, the nascent CFTR has been shown to interact with other cytoplasmic chaperones in addition to the major Hsp70- and Hsp90-based systems, and it is con- ceivable that their manipulation might also augment maturation of the mutant CFTR.

Examples include the Hdj-2-related cysteine- string protein, which seems to interact with both the ER and post-Golgi forms of the CFTR (106), and small Hsps, including αA- crystallin, which may contribute to ERAD of the misfolded CFTR (120).

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Multiple cytoplasmic chaperones and cochaperones act coordinately and sequen- tially in appraising and influencing the as- sembly of the large CFTR domains at the cytoplasmic surface of the ER; however, the influence of luminal ER chaperones is less clear. Both calnexin and calreticulin bind the core N-linked oligosaccharide chains on the fourth extracytoplasmic loop (115) as does EDEM (107). Although these lectin chaper- ones together with key glycosidases and gly- cosyl transferases constitute a conformation- sensing mechanism that determines the fate of many glycoproteins during biogenesis (109), this is apparently not a rate-limiting pathway for the CFTR. This can be concluded be- cause the wild-type protein can be exported from the ER entirely without glycosylation, whereas unglycosylated F508 still cannot

(121).

Nevertheless, there have been several at- tempts to promote “release” of the mutant from the ER by perturbing interactions with the membrane-bound calnexin. Because it and its luminal counterpart, calreticulin, are calcium-dependent chaperones, thapsigarin treatment to deplete ER calcium was reported to cause some maturation (122) as was another agent, curcumin (123). Although the latter compound may interact with the CFTR (48, 124) as with many other proteins, an ability to promote F508 maturation and stability has not been generally confirmed (125) nor has an influence on the interaction between calnexin and the nascent CFTR (126). Knockdown of calnexin by RNAi also did not elicit F508 maturation (127). Calreticulin, which is trans- ported from the ER to the cell surface, has been reported to promote CFTR transport to that location where it is rapidly endocytosed and degraded (128). Even though the luminally exposed loops between membrane-spanning sequences of the CFTR are quite short, additional ER lu- minal chaperones including GRP78, GRP75, reticulocalbin, and calumenin were iden- tified in immunoprecipitates from CFTR-

expressing cells (116). These interactions and their possible roles of nascent CFTR process- ing remain to be explored further.

Traffic in Endocytic and Distal Secretory Pathways

The population of wild-type CFTR molecules that are exported from the ER and reach the Golgi and post-Golgi compartments is quite stable with a half-life of approximately 16 h (90, 91). However, the cell surface CFTR pool was very rapidly internalized at a rate of approximately 10% per minute (129). Because the biosynthetic rate is very much less than that, this indicated that the internalized protein must be recycled to the surface. This was shown to be the case in various cell types using different methodologies (130, 131). The “in” route has been extensively characterized and appears to follow, at least primarily, the clathrin-coated vesicle (CCV) endocytic pathway (132). The “out” route is beginning to be better under- stood (see below). The CFTR was detected in isolated CCVs (133) and visualized in endo- somes by immunocytochemistry (134). The major functional implications of this entry and exit cycle are with respect to regulation of the amounts of cell surface CFTR activity and the fact that it is strongly perturbed by the F508 mutation. Lukacs and coworkers (132) observed a phosphorylation influence on clathrin-dependent CFTR endocytosis, and some investigations have emphasized the role of a shift in the balance between the plasma membrane and intracellular vesicular pools by PKA phosphorylation (135). However, the extent to which this mechanism contributes to epithelial PKA- stimulated chloride secretion compared to the activation of individual CFTR channels already in the plasma membrane is not yet clear. What is clear is that when F508 and other processing variants of the CFTR have reached the plasma membrane, either in cells

www.annualreviews.org CFTR Function and Prospects for Therapy

CCV: clathrin-

coated vesicle

713

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

grown at reduced temperature or because of genetic or pharmacological manipulation, they are thermally unstable (111, 136) and again very rapidly removed from the surface (137). This reduction in surface steady-state amount has been attributed to accelerated endocytosis in one study (138) and to fail- ure of recycling in another (131). The differ- ence may reflect the different cell types used, but it will be important to resolve this is- sue in the fully differentiated epithelial cells of the tissues most affected by the disease. Binding of tyrosine- and dileucine-based sig- nals in the CFTR C-terminal extension to the μ 2 subunit of the AP-2 clathrin adap- tor complex initiates clathrin-mediated endo- cytosis (139). Notwithstanding the fact that F508 and other mutations impacting CFTR structure cause enhanced clearance from the cell surface, introduction of endocytic motifs elsewhere in the protein than the C termi- nus apparently can also augment endocytosis

(140).

In addition to trafficking between the plasma membrane, early endosomes, and re- cycling endosomes, the CFTR also may pass into late endosomes, multivesicular bodies, and lysosomes for degradation, and indeed the latter routing is the ultimate fate of the endocytosed F508 CFTR (141). Details of the recognition mechanisms that are able to distinguish the wild-type from the mutant CFTR and thereby determine the directions taken are not yet entirely understood, but the Lukacs laboratory (131) has demonstrated the involvement of ubiquitylation and other fac- tors that participate in routing of the ubiqui- tylated substrate. This ubiquitylation and its recognition appear responsible for the mu- tant protein proceeding to lysosomal degrada- tion, using an Rme-1-dependent mechanism, rather than being recycled like the wild type

(130).

The actin-based cytoskeleton plays a ma- jor role in the endocytic trafficking of the CFTR as it does of other internalized cargo. Myosin VI participates in endocytic uptake, perhaps by providing an actin-binding mo-

714

Riordan

tor to drive directional movement of CCVs (142), whereas myosin Vb may act in an analogous fashion in trafficking from recy- cling endosomes back to the plasma mem- brane (143). In both cases, filamentous actin assemblies are required. Disruption of these assemblies by several different means has recently been shown to influence both inter- nalization and recycling of the CFTR (144). However, the net effect was a large reduc- tion in the size of the cell surface CFTR pool, apparently because of augmented inter- nalization. The later effect was interpreted as the result of freeing of a cytoskeletally constrained plasma membrane pool, which could be recruited more readily into clathrin- coated pits (144). It is not clear how to rec- oncile this increased internalization on dis- ruption of the actin cytoskeleton if it is required in the endocytosis-promoting action of myosin VI (142). Populations of CFTR molecules constrained in their mobility have been demonstrated in independent studies us- ing different methodologies (145, 146). Con- nections of the CFTR to the actin cytoskele- ton may be mediated by both PDZ-domain proteins binding at the C terminus (147) and filamin at the N terminus (148). Internaliza- tion and transport to lysosomes for degrada- tion are promoted when filamin binding is prevented. The entire significance of the extensive trafficking and scrutiny of the CFTR in the endocytic and post-Golgi compartments may not yet be fully appreciated. It clearly provides additional mechanisms of quality control be- yond the apparently very stringent ones ap- plied at the ER during synthesis and assembly. There is evidence that the mutant CFTR can impact the trafficking of other cellular con- stituents, including lipids, if it gains access to the distal compartments (149). However, distal quality control may provide an elab- orate mechanism to control the turnover of the mature ion channel. Perhaps, changes in the molecule, which may occur as it ages in an environment probably not perfectly pro- tected from damaging chemical species, are

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

continuously monitored for disposal. In this way, either genetic or environmental per- turbations would be similarly handled by a highly evolved mechanism, probably sharing features with systems that distinguish occu- pied and unoccupied cell surface receptors

(150).

PROSPECTS FOR THERAPY

In addition to advancing the understanding of several aspects of human genetics and the molecular and cellular biology of epithelial ion and fluid homeostasis, knowledge of the CFTR gene and protein has provided new opportunities to develop rational therapeu- tic approaches to the treatment of CF. Those currently in progress include CFTR gene re- placement, suppression of nonsense muta- tions, restoration of folding and function of mutant CFTR channels, activation of alter- native chloride channels, inhibition of sodium absorption, and other ionic and osmotic ma- nipulations to normalize fluid and bicarbon- ate levels on epithelial surfaces. These newer strategies are intended to complement current treatments, which combat mucus viscosity and bacterial infection (151, 152).

CFTR Gene Replacement

Efforts to develop gene replacement ther- apy for CF have been ongoing since shortly after the gene was discovered. Both non- replicating viral vectors and DNA-lipid com- plexes have been employed but without defini- tive evidence of persistent functional efficacy. However, substantial progress has been made in understanding the limitations to the suc- cess of the early attempts. The target of the aerosolized vector is the apical surface of air- way and submucosal gland epithelial cells. The lack of receptors for adenovirus or adeno- associated virus serotypes, initially employed, precluded significant gene transfer into these target cells. However, there are receptors for alternative adeno-associated virus (AAV)

serotypes, and these have been employed for delivery of CFTR cDNA to cultured cells and the airways of mice and macaques (153, 154). The limited size of the AAV genome also constrains the size of the expression cassette that can be incorporated. Both the CFTR cDNA and enhancer/promoter elements have been minimized. In one approach, N-terminal truncations of the CFTR cDNA, removing either the first 117 or 264 amino acids of the protein, were employed (154). Although these N-terminally truncated CFTRs appear capable of generating chloride channel ac-

tivity (155), their ability to mature and traf- fick to the surface of human cells is expected to be compromised. In an alternative strat- egy, in addition to minimizing the length of

a CMV promoter, an intron and an interven-

ing sequence element, a short region of the R domain not essential to maturation or func- tion, were deleted (153). Exposure to AAV5 used for packaging of this construct imparted

transepithelial chloride transport on CF pri- mary epithelial cell cultures. These and other improvements to AAV- based vectors provide hope that greater clini- cal benefit may occur in future clinical trials. Although host immune response to AAV vec- tors appears to be less of a problem than with other viral gene transfer vectors, Limberis and colleagues (156) have recently observed

a T cell response to the CFTR protein it-

self in mice to which CFTR cDNA was de- livered with a viral vector. Although there is likely to be tolerance to the CFTR in most patients who are candidates for gene ther- apy, this would presumably not be the case with infrequent homozygous null mutations; hence, CFTR-specific T cell responses would need to be monitored in such cases. Still of more immediate concern, however, are prac- tical issues of efficient delivery and persistent expression in a setting in which pathological changes, including inflammation and infec- tion, probably have already occurred. These issues still need to be effectively dealt with in parallel with the ongoing improvements and innovation in vector development (157).

www.annualreviews.org CFTR Function and Prospects for Therapy

715

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

716

Suppression of Premature Stop Mutations

Like gene replacement therapy, promotion of read-through translation of transcripts with nonsense mutations is a conceptually simple approach to therapy for genetic diseases, such as CF. Nearly 10% of patients possess an al- lele with a premature stop codon in the CFTR coding sequence. However, in some popula- tions, such as of those of Ashkenazi Jewish origin, this is the predominant type of mu- tation. The lack of CFTR protein generally results in a severe disease phenotype (158). Amino glycoside antibiotics can enable low- frequency insertion of an amino acid at the po- sition of the stop codon, resulting in synthesis of a small amount of full-length protein (159). This has been observed to occur to some ex- tent in cultured cells (160) and in transgenic mice expressing common CF-associated stop mutations (161). Gentamycin application directly to the nasal mucosa of such patients also yielded en- couraging results, causing a significant de- crease in transepithelial potential difference and an apparent increase in the full-length CFTR as detected by immunocytochemical staining (162). This study focused on a single stop mutation common in the Israeli popula- tion (W1282X), and the nasal potential dif- ference measurements, all made at a single center, were statistically quite uniform. In- terestingly, this truncation in the middle of NBD2 is compatible with conformational maturation and a low level of chloride channel function (47, 163). A more recent multicen- ter study of patients with different nonsense mutations did not detect significant improve- ments of nasal potential difference or changes in the amount or localization of CFTR pro- tein in similarly treated nasal epithelial cells (164). There were several technical differ- ences in the two studies that could contribute to the apparently disparate results, but the outcome is suggestive that truncations remov- ing more than just NBD2 from the protein may be less responsive to these aminoglyco-

Riordan

sides. However, effectiveness even in patients with the W1282X mutation alone would rep- resent a highly significant accomplishment if long-term delivery of an appropriate com- pound, producing clinical benefit without ma- jor side effects, can be achieved. This ap- proach is illustrative of the likely evolution of patient- or population-specific therapies for CF where different treatments will be effec- tive for different genotypes.

Restoration of CFTR Folding and Function

Because F508- and other CFTR-processing mutant proteins are synthesized and ca- pable of functioning under certain condi- tions, there are intensive efforts to identify small molecules that can promote process- ing. High-throughput screens have employed assays that measure CFTR-mediated halide efflux from cells using a halide-sensitive flu- orescent protein (165) or CFTR-dependent changes in membrane potential (166). A strat- egy employed by Verkman and collaborators (167) has proceeded through several stages and proven very effective thus far. In the first stages, both inhibitors (168) and acti- vators (169) of the wild-type CFTR chan- nel activity were identified. In addition to verifying the utility of the assay for screen- ing, the two classes of inhibitors discovered may potentially block the chronically acti- vated CFTR in secretory diarrheas (168) and also help confirm that membrane permeabil- ity changes, caused by positively acting mod- ulators of the mutant CFTRs, are actually occurring through CFTRs. The two types of inhibitors apparently have different modes of action: Thiazolidinones cause a greatly pro- longed channel closed state (168), and glycine hydrazides (170) strongly reduce the mean open time. Cells expressing the F508 CFTR were first screened using the same assay for so- called potentiators, compounds that could in- crease the activity of the mutant channel, par- tially rescued by culture at low temperature

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

(171, 172). Different classes of effective com- pounds able to activate several disease-causing mutants as well as F508 were identified, in- cluding tetrahydrobenzothiophenes, sulfon- amides, and phenylglycines. In the highly sig- nificant next step, F508 CFTR-expressing cells were exposed to test compounds for longer periods (18 h) during which suf- ficient co- and posttranslational processing, transport to, and accumulation at the cell surface could occur (89). Several classes of compounds, achieving this end at micro- molar concentrations or less, were found. Among these were bisaminomethylbithia- zoles, aminoarylthiazoles, and quinazoliny- laminopyrimidinones, which over a period of several hours have a similar effect as growth of cells at 27 C. Rescue was confirmed by the appearance of CFTR-mediated current and mature proteins at the cell surface. Some cor- rective effect was detected in differentiated bronchial epithelial cells expressing F508. Structure-activity relationship studies and op- timization of the lead compounds and scaf- folds identified should yield compounds ap- propriate for clinical trials. A parallel screening and drug development program by another group has also yielded both potentiator and corrector compounds (166). A quinazolinone scaffold and deriva- tives from the later group have been fairly ex- tensively characterized in cell systems (173). One of these, VRT-325, causes partial matu- ration of F508 and some other misprocess- ing mutants, but is nonspecific, also acting on the related P-glycoprotein multidrug trans- porter (174) and unrelated hERG potassium channels (166). It is also highly hydropho- bic and toxic to cells. However, additional compounds more suitable for optimization as drug candidates are in development. A pyra- zole compound, VRT-532, also identified in this program, potentiated the activity of res- cued F508. Thus, the progress of these two groups has provided proof of principle and validated approaches for the discovery of small molecules to combat misprocessing and dys-

function of the mutant CFTRs by employ- ing high-throughput cell-based screening of CFTR function. Recently, additional com- pounds, able to promote the appearance of the F508 CFTR protein at the cell surface, have also been reported (175), as have a va- riety of compounds selected arbitrarily or in small biased screens (176). The modes of action of the effective po- tentiators and correctors identified so far have not yet been established, and there is no di- rect evidence that they interact directly with the CFTR. However, interesting observations from the Clarke laboratory (177) have indi- cated that some pairs of the compounds have additive corrective effects, suggesting differ- ent sites of action. Furthermore, one mem- ber of this corrector pair was originally iden- tified as a potentiator, indicating that some agents may have both effects. Together with its specificity of action on the CFTR, but not on P-glycoprotein, this provides suggestive evidence of direct binding to the CFTR. Not surprisingly, many of the small molecules turned up in corrector screens are very hydrophobic, having entered cells to gain access to the ER. As mentioned, the VRT-325 corrector has this property, making it fairly toxic to cells and nonspecific as well as in- fluencing other membrane proteins. Never- theless, its apparent action on the MSDs of the CFTR is quite informative (178), con- sistent with the facts that the F508 muta- tion in NBD1 disrupts folding of the MSDs (47, 99) and that rescue can be achieved by overcoming this effect. By contrast, at least some potentiators have been reported to act at the level of the cytoplasmic NBDs (179). Such agents if able to repair the functional defect caused by an NBD mutation, such as F508, and in so doing also prevent the sec- ondary MSD disruption, might be preferable to membrane active agents for several reasons. The main disadvantage of the latter group, in addition to their possible toxicity and lack of selectivity, could be that while circumvent- ing quality control they may leave functional

www.annualreviews.org CFTR Function and Prospects for Therapy

717

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

718

regulation uncorrected. Moreover, because there are redundant quality control systems detecting the state of both membrane and cy- toplasmic domains (99), it will be necessary to correct changes in both with individual or combined agents. Although there are suggestions that small amounts of F508 protein may normally

reach the cell surface in some patients (180),

it is unlikely that this is sufficient to pro-

vide normal function even if it were fully ac- tive. Therefore, most effort is appropriately focused on overcoming ER retention and getting misprocessed mutants to the plasma membrane. However in addition to a kinetic defect in folding and assembly, the Phe508 deletion results in a thermally unstable pro- tein (111, 136). Both the functional and bio- chemical half-lives of the rescued mutant

protein are greatly shortened. Thus, ideal cor- rective compounds need to stabilize the pro- tein as well as restore folding and function.

It is known that the folding yield and sub-

sequent stability of CFTR variants are not strictly coupled (108) and that the VRT-325 corrector caused only a partial extension of the lifetime of the F508 protein that it rescued (166). Another corrector appeared unable to stabilize the surface mutant protein that it had freed from ER retention (89). Cellular manip- ulations, such as inhibition of endocytosis, are

capable of stabilizing the F508 CFTR at the cell surface (181), but these types of unspecific manipulations are impractical therapeutically. To be entirely effective, chemical agents indi- vidually, or in combination, will have to cor- rect folding, assembly, function, and stability

in a selective and nontoxic manner. Although

a tall order, this now appears to be within

the combined drug development capabilities of academic and industrial laboratories.

Modulation of Other Epithelial Ion Transport Pathways

The CFTR provides a crucial regulatory step

in epithelial anion (Cl , HCO 3 , and perhaps

others) permeability. Its complex regulatory

Riordan

properties enable it to gauge the level of secre- tion or reabsorption required in different ep- ithelial tissues. However, there are additional anion channels in at least some of these epithe- lia, which can and do contribute to secretion. For example in the crucial airways, calcium- activated chloride channels, which can be acti- vated by purinergic receptors, provide poten- tial alternative pathways to the CFTR (182, 183). Nucleotide analogue agonists of the coupled P2Y 2 receptors are currently in clini- cal trials (184). Also contributing to the debil- itating dehydration of the airway surface is the fact that Na + absorption continues unabated in the face of inadequate Cl secretion (72). Therefore, Na + channel inhibitors have the potential to ameliorate this situation, and the classic ameloride inhibitor has been observed to do this in the short term (185). More po- tent and longer-lived ENaC inhibitors have been developed and are also in clinical trials (186). Thus, judicious dosing with alternative anion channel activators and ENaC inhibitors or other agents that intercede in the biochem- ical and cellular pathways that control them may be able to partially mimic the role of the CFTR. The feasibility of such non-CFTR-based approaches is illustrated by the effects of adding osmotically active agents to the air- ways (187). The increased water that follows osmotically with the addition of hypertonic saline causes increased mucociliary clearance and improved lung function (188). Although restoration of normal CFTR function by any means is clearly the ultimate objective, these alternative methods of generating a more nor- mally balanced epithelial surface hydration also offer extremely important approaches to combating CF lung disease.

CONCLUSIONS AND OUTLOOK

The CFTR has provided a focus for an ever broadening array of studies of CF and a spec- trum of less severe “CFTR-opathies.” Much has been learned, but there remain many cru- cial unresolved issues. A frequently expressed

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

frustration in the field is that defects in the primary function of the CFTR do not seem to link simply to the myriad pathophysio- logical features of the disease. This some- times leads to proposals that the protein must have alternate or additional functions. De- termining whether this is the case, or if the linkages with downstream effects have not yet been dissected out, can be quite chal- lenging. Cl is quantitatively the predomi- nant permeant anion. Yet HCO 3 is of great importance, and its role in mucin process- ing is under intense investigation. Other an- ions, including SCN , may be important in the innate immunity of the airway. The mechanisms underlying the interplay between CFTR- and ENaC-mediated Na + permeabil- ity in airway epithelia have yet to be re- solved and are necessary to fully understand how ion and liquid homeostasis is maintained there. The CFTR protein itself has revealed un- expected and fascinating properties, but its mechanism of action remains incompletely understood. This is partly because of diffi- culties in reproducibly obtaining large sta- ble preparations of purified and reconstituted protein for rigorous characterization of its enzymology, channel activity, and physical properties. The 3D structure at atomic reso- lution must overcome several technical chal- lenges. However, the resolution obtained by electron crystallography of both the wild-type

and important variants such as F508 will be improved and, in combination with bioin- formatic and computational methods, yield useful structural models. The focus of these models on alterations in intermediate folding states or perturbed interdomain associations may enable rational structure-based drug de- sign. Because both the dynamics and disorder inherent in the CFTR are among the imped- iments to X-ray crystallography, it is likely that continued major advances will be made in NMR studies of the type that have recently provided deeper insight into the action of the unique R domain. As a firmly established therapeutic target in CF (and a spectrum of less severe clini- cal conditions) mutant CFTR correction can be pursued by all available experimental high- throughput strategies such as whole-genome siRNA and others. The recent demonstration that quantitative downregulation of a specific Hsp 90 cochaperone could provide partial res- cue of F508 provides encouragement that additional subtle manipulations of the nascent CFTR chaperome may provide a means for generation of sufficient mature protein. Cur- rently, there are indications of a possible re- naissance in gene therapy efforts in CF, and significant advances may be anticipated. The way forward is persistence in the pursuit of the defective mechanisms that must be over- come or supplanted to alleviate the symptoms of this disease.

SUMMARY POINTS

1. Although influenced by other environmental and genetic factors, CF may still be considered a single-gene defect disease. Therefore, understanding and restoration of the function of the CFTR are reasonable approaches to therapy.

2. As an ion channel, the CFTR’s distinguishing feature is its ability to hydrolyze its bound ligand rather than respond to changes in bulk ligand concentrations as is done by all other known ligand-gated channels.

3. Essential for utilization of the basic ABC transporter architecture as an ion channel is the precisely graded control of gating by the phosphorylation state of multiple sites in the unique disordered R domain.

www.annualreviews.org CFTR Function and Prospects for Therapy

719

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

4. A large number of known mutations have been detected in CF patients, so therapeutic strategies can be tailored to these mutation types.

5. As a complex multidomain membrane protein, the CFTR is assembled inefficiently during biosynthesis, and many mutations, including F508, present in 90% of CF patients, are unable to mature conformationally and succumb to quality control mechanisms in the ER and endocytic compartments.

6. First generations of small molecules that can rescue these processing mutants and augment channel function have been identified, and extensive efforts are being devoted to further the discovery and development of effective pharmaceuticals.

7. There is also ongoing progress in gene replacement methodologies, which will be effective for all mutation types, and treatments to suppress nonsense mutations and manipulate other ion transport pathways, which influence hydration of epithelial sur- faces to either mimic the CFTR or compensate for its absence.

FUTURE ISSUES

1. The molecular mechanism of action of CFTR is still far from completely resolved. The specific features of the protein that enable it to operate as an ion channel while other proteins, of generally similar structure, perform active transport need to be clarified. How similar or different are the allosteric coupling events between NBDs and MSDs that drive active transport and those that determine the gating state of the CFTR channel? What is the molecular structure of the CFTR anion selective pore? How does the unique R domain provide overriding control of the basic ABC transporter mechanism? High-resolution 3D structures of the CFTR protein at dif- ferent stages of its catalytic and gating cycles as well as different phosphorylation states will be necessary, but not entirely sufficient, to answer these questions. The dy- namic structural changes underlying functional transitions will have to be discerned from complementary approaches including kinetic analyses of CFTR’s enzymatic and single-channel activities as well as spectroscopic and computational methods.

2. Success in the development of molecular therapies for CF will require new break- throughs in gene replacement technology, further elucidation of the misfolding and missassembly of F508 CFTR, as well as the cellular mechanisms that recognize the mutant protein and determine its fate. In addition, there is a need for further progress in understanding and manipulating major phenotypic modifications downstream from mutant CFTR, particularly alterations in the physical properties of mucins.

DISCLOSURE STATEMENT

J.R.R. has commercial interest in a company targeting mutant CFTRs.

ACKNOWLEDGMENT

The author’s laboratory is supported by grants from the NIH and the Cystic Fibrosis Foun- dation. I thank members of the Riordan laboratory for critical reading of the manuscript and Anne Edwards for its assembly.

720

Riordan

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

LITERATURE CITED

1.

Davis PB. 2006. Am. J. Respir. Crit. Care Med. 173:475–82

2.

Quinton PM. 2007. Physiology 22:212–25

3.

Accurso FJ. 2007. Am. J. Respir. Crit. Care Med. 175:754–57

4.

Higgins CF, Haag PD, Nikaido K, Ardeshir F, Garcia G, Ames GF. 1982. Nature

298:723–27

5.

Gerlach JH, Endicott JA, Juranka PF, Henderson G, Sarangi F, et al. 1986. Nature

324:485–89

6.

Gros P, Croop J, Housman D. 1986. Cell 47:371–80

7.

Higgins CF, Hiles ID, Whalley K, Jamieson DJ. 1985. EMBO J. 4:1033–40

8.

Felmlee T, Pellett S, Welch RA. 1985. J. Bacteriol. 163:94–105

9.

Holland B, Higgins CF, Kuchler K, Cole SPC, eds. 2003. ABC Proteins: From Bacteria to Man. New York: Elsevier Sci.

10.

Dean M. 2005. Methods Enzymol. 400:409–29

11.

Aleksandrov L, Aleksandrov AA, Chang XB, Riordan JR. 2002. J. Biol. Chem. 277:15419–

25

12.

Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, et al. 2000. Cell 101:789–800

13.

Karpowich N, Martsinkevich O, Millen L, Yuan YR, Dai PL, et al. 2001. Structure 9:571–

86

14.

Moody JE, Millen L, Binns D, Hunt JF, Thomas PJ. 2002. J. Biol. Chem. 277:21111–14

15.

Smith PC, Karpowich N, Millen L, Moody JE, Rosen J, et al. 2002. Mol. Cell 10:139–49

16.

Chen J, Lu G, Lin J, Davidson AL, Quiocho FA. 2003. Mol. Cell 12:651–61

17.

Zaitseva J, Jenewein S, Wiedenmann A, Benabdelhak H, Holland IB, Schmitt L. 2005.

Biochemistry 44:9680–90

18.

Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, et al. 2006. EMBO J.

25:3432–43

19.

Locher KP, Lee AT, Rees DC. 2002. Science 296:1091–98

20.

Dawson RJ, Locher KP. 2006. Nature 443:180–85

21.

Pinkett HW, Lee AT, Lum P, Locher KP, Rees DC. 2007. Science 315:373–77

22.

Hollenstein K, Frei DC, Locher KP. 2007. Nature 446:213–16

23.

Rosenberg MF, Callaghan R, Modok S, Higgins CF, Ford RC. 2005. J. Biol. Chem.

280:2857–62

24.

Rosenberg MF, Callaghan R, Modok S, Higgins CF, Ford RC. 2005. J. Biol. Chem.

280:2857–62

25.

Awayn NH, Rosenberg MF, Kamis AB, Aleksandrov LA, Riordan JR, Ford RC. 2005. Biochem. Soc. Trans. 33:996–99

26.

Lewis HA, Buchanan SG, Burley SK, Conners K, Dickey M, et al. 2004. EMBO J.

23:282–93

27.

Lewis HA, Zhao X, Wang C, Sauder JM, Rooney I, et al. 2005. J. Biol. Chem. 280:1346–53

28.

Hegedus T, Riordan JR. 2006. Cent. Euro. J. Biol. 1:29–42

29.

Dulhanty AM, Riordan JR. 1994. FEBS Lett. 343:109–14

30.

Dulhanty AM, Riordan JR. 1994. Biochemistry 33:4072–79

31.

Ostedgaard LS, Baldursson O, Vermeer DW, Welsh MJ, Robertson AD. 2000. Proc. Natl. Acad. Sci. USA 97:5657–62

32.

Baker JM, Hudson RP, Kanelis V, Choy WY, Thibodeau PH, et al. 2007. Nat. Struct. Mol. Biol. 14:738–45

33.

Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, et al. 1989. Science 245:1066–

73

www.annualreviews.org CFTR Function and Prospects for Therapy

721

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

34. Kunzelmann K. 2001. News Physiol. Sci. 16:167–70

35. Lee MG, Wigley WC, Zeng W, Noel LE, Marino CR, et al. 1999. J. Biol. Chem.

274:3414–21

36. Venglarik CJ, Schultz BD, Frizzell RA, Bridges RJ. 1994. J. Gen. Physiol. 104:123–46

37. Zou X, Hwang TC. 2001. Biochemistry 40:5579–86

38. Hanrahan JW, Wioland MA. 2004. Proc. Am. Thorac. Soc. 1:17–21

39. Hanrahan JW, Kone Z, Mathews CJ, Luo J, Jia Y, Linsdell P. 1998. Methods Enzymol.

293:169–94

40. Hanrahan JW, Tabcharani JA, Becq F, Mathews CJ, Augustinas O, et al. 1995. Soc. Gen. Physiol. Ser. 50:125–37

41. Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, Bridges RJ. 1999. J. Gen. Physiol. 113:743–60

42. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. 2001. Nature 410:94–

97

43. Coakley RD, Grubb BR, Paradiso AM, Gatzy JT, Johnson LG, et al. 2003. Proc. Natl. Acad. Sci. USA 100:16083–88

44. Krouse ME, Talbott JF, Lee MM, Joo NS, Wine JJ. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 287:L1274–83

45. Li C, Ramjeesingh M, Wang W, Garami E, Hewryk M, et al. 1996. J. Biol. Chem.

271:28463–68

46. Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC. 2006. EMBO J.

25:4728–39

47. Cui L, Aleksandrov L, Chang XB, Hou YX, He L, et al. 2007. J. Mol. Biol. 365:981–94

48. Wang W, Bernard K, Li G, Kirk KL. 2007. J. Biol. Chem. 282:4533–44

49. Grimard V, Li C, Ramjeesingh M, Bear CE, Goormaghtigh E, Ruysschaert JM. 2004. J. Biol. Chem. 279:5528–36

50. Anderson MP, Berger HA, Rich DP, Gregory RJ, Smith AE, Welsh MJ. 1991. Cell

67:775–84

51. Baukrowitz T, Hwang TC, Nairn AC, Gadsby DC. 1994. Neuron 12:473–82

52. Gunderson KL, Kopito RR. 1995. Cell 82:231–39

53. Ishihara H, Welsh MJ. 1997. Am. J. Physiol. 273:C1278–89

54. Hennager DJ, Ikuma M, Hoshi T, Welsh MJ. 2001. Proc. Natl. Acad. Sci. USA 98:3594–99

55. Aleksandrov AA, Riordan JR. 1998. FEBS Lett. 431:97–101

56. Aleksandrov AA, Chang X, Aleksandrov L, Riordan JR. 2000. J. Physiol. 528(Part 2):259–

65

57. Vergani P, Nairn AC, Gadsby DC. 2003. J. Gen. Physiol. 121:17–36

58. Berger AL, Ikuma M, Welsh MJ. 2005. Proc. Natl. Acad. Sci. USA 102:455–60

59. Vergani P, Lockless SW, Nairn AC, Gadsby DC. 2005. Nature 433:876–80

60. Csanady L, Nairn AC, Gadsby DC. 2006. J. Gen. Physiol. 128:523–33

61. Aleksandrov AA, Aleksandrov LA, Riordan JR. 2007. Pfl¨ugers Arch. 453:693–702

62. Locher KP. 2004. Curr. Opin. Struct. Biol. 14:426–31

63. Rosenberg MF, Kamis AB, Aleksandrov LA, Ford RC, Riordan JR. 2004. J. Biol. Chem.

279:39051–57

64. Ketchum CJ, Rajendrakumar GV, Maloney PC. 2004. Biochemistry 43:1045–53

65. Randak C, Welsh MJ. 2003. Cell 115:837–50

66. Gross CH, Abdul-Manan N, Fulghum J, Lippke J, Liu X, et al. 2006. J. Biol. Chem.

281:4058–68

67. Randak C, Neth P, Auerswald EA, Eckerskorn C, Assfalg-Machleidt I, Machleidt W. 1997. FEBS Lett. 410:180–86

722

Riordan

68.

Bhaskara V, Dupre A, Lengsfeld B, Hopkins BB, Chan A, et al. 2007. Mol. Cell 25:647–61

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

69.

Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, et al. 1995. Science 269:847–50

70.

Boucher RC. 2007. J. Intern. Med. 261:5–16

71.

Boucher RC. 2007. Trends Mol. Med. 13:231–40

72.

Mall M, Grubb BR, Harkema JR, O’Neal WK, Boucher RC. 2004. Nat. Med. 10:487–93

73.

Gabriel SE, Clarke LL, Boucher RC, Stutts MJ. 1993. Nature 363:263–68

74.

Di A, Brown ME, Deriy LV, Li C, Szeto FL, et al. 2006. Nat. Cell Biol. 8:933–44

75.

Painter RG, Valentine VG, Lanson NA Jr, Leidal K, Zhang Q, et al. 2006. Biochemistry

45:10260–69

76.

Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DWH. 1995. Am. J. Respir. Crit. Care Med. 151:1075–82

77.

Pier GB, Grout M, Zaidi TS, Olsen JC, Johnson LG, et al. 1996. Science 271:64–67

78.

Kowalski MP, Dubouix-Bourandy A, Bajmoczi M, Golan DE, Zaidi T, et al. 2007. Science

317:130–32

79.

Moskwa P, Lorentzen D, Excoffon KJ, Zabner J, McCray PB Jr, et al. 2007. Am. J. Respir. Crit. Care Med. 175:174–83

80.

Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, et al. 1989. Science

245:1073–80

81.

Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, et al. 1990. Cell 63:827–34

82.

Kartner N, Augustinas O, Jensen TJ, Naismith AL, Riordan JR. 1992. Nat. Genet. 1:321–

27

83.

Mall M, Kreda SM, Mengos A, Jensen TJ, Hirtz S, et al. 2004. Gastroenterology 126:32–41

84.

Kreda SM, Mall M, Mengos A, Rochelle L, Yaskaskas J, et al. 2005. Mol. Biol. Cell 16:2154–

67

85.

Drumm ML, Wilkinson DJ, Smit LS, Worrell RT, Strong TV, et al. 1991. Science

254:1797–99

86.

Li C, Ramjeesingh M, Reyes E, Jensen T, Chang X-B, et al. 1993. Nat. Genet. 3:311–16

87.

Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ. 1992. Nature

358:761–64

88.

Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ. 1996. Cell Stress Chap- erones 1:117–25

89.

Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, et al. 2005. J. Clin. Investig.

115:2564–71

90.

Ward CL, Kopito RR. 1994. J. Biol. Chem. 269:25710–18

91.

Lukacs GL, Mohamed A, Kartner N, Chang X-B, Riordan JR, Grinstein S. 1994. EMBO J. 13:6076–86

92.

Gelman MS, Kopito RR. 2003. Methods Mol. Biol. 232:27–37

93.

Ward CL, Omura S, Kopito RR. 1995. Cell 83:121–27

94.

Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR. 1995. Cell 83:129–

35

95.

Sato S, Ward CL, Kopito RR. 1998. J. Biol. Chem. 273:7189–92

96.

Loo MA, Jensen TJ, Cui L, Hou Y, Chang XB, Riordan JR. 1998. EMBO J. 17:6879–87

97.

Loo TW, Bartlett MC, Clarke DM. 2004. J. Biol. Chem. 279:38395–401

98.

Baldursson O, Ostedgaard LS, Rokhlina T, Cotten JF, Welsh MJ. 2001. J. Biol. Chem.

276:1904–10

99.

Younger JM, Chen L, Ren HY, Rosser MF, Turnbull EL, et al. 2006. Cell 126:571–82

100.

Wang X, Matteson J, An Y, Moyer B, Yoo JS, et al. 2004. J. Cell Biol. 167:65–74

101.

Pagant S, Kung L, Dorrington M, Lee MC, Miller EA. 2007. Mol. Biol. Cell 18:3398–413

www.annualreviews.org CFTR Function and Prospects for Therapy

723

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

102. Vij N, Fang S, Zeitlin PL. 2006. J. Biol. Chem. 281:17369–78

103. Cyr DM. 2005. Nat. Struct. Mol. Biol. 12:2–3

104. Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM. 2001. Nat. Cell Biol. 3:100–

5

105. Meacham GC, Lu Z, King S, Sorscher E, Tousson A, Cyr DM. 1999. EMBO J. 18:1492–

505

106. Zhang H, Schmidt BZ, Sun F, Condliffe SB, Butterworth MB, et al. 2006. J. Biol. Chem.

281:11312–21

107. Gnann A, Riordan JR, Wolf DH. 2004. Mol. Biol. Cell 15:4125–35

108. Du K, Sharma M, Lukacs GL. 2005. Nat. Struct. Mol. Biol. 12:17–25

109. Parodi AJ. 2000. Annu. Rev. Biochem. 69:69–93

110. Thibodeau PH, Brautigam CA, Machius M, Thomas PJ. 2005. Nat. Struct. Mol. Biol.

12:10–16

111. Cui L, Aleksandrov L, Hou YX, Gentzsch M, Chen JH, et al. 2006. J. Physiol. 572:347–58

112. Zhang F, Kartner N, Lukacs GL. 1998. Nat. Struct. Biol. 5:180–83

113. Seibert FS, Loo TW, Clarke DM, Riordan JR. 1997. J. Bioenerg. Biomembr. 29:429–42

114. Yang Y, Janich S, Cohn JA, Wilson JM. 1993. Proc. Natl. Acad. Sci. USA 90:9480–84

115. Pind S, Riordan JR, Williams DB. 1994. J. Biol. Chem. 269:12784–88

116. Wang X, Venable J, LaPointe P, Hutt DM, Koulov AV, et al. 2006. Cell 127:803–15

117. Alberti S, Bohse K, Arndt V, Schmitz A, Hohfeld J. 2004. Mol. Biol. Cell 15:4003–10

118. Choo-Kang LR, Zeitlin PL. 2001. Am. J. Physiol. Lung Cell Mol. Physiol. 281:L58–68

119. Jiang C, Fang SL, Xiao YF, O’Connor SP, Nadler SG, et al. 1998. Am. J. Physiol.

275:C171–78

120. Ahner A, Nakatsukasa K, Zhang H, Frizzell RA, Brodsky JL. 2007. Mol. Biol. Cell 18:806–

14

121. Marshall J, Fang S, Ostedgaard LS, O’Riordan CR, Ferrara D, et al. 1994. J. Biol. Chem.

269:2987–95

122. Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, et al. 2002. Nat. Med.

8:485–92

123. Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, et al. 2004. Science 304:600–2

124. Berger AL, Randak CO, Ostedgaard LS, Karp PH, Vermeer DW, Welsh MJ. 2005. J. Biol. Chem. 280:5221–26

125. Loo TW, Bartlett MC, Clarke DM. 2004. Biochem. Biophys. Res. Commun. 325:580–85

126. Grubb BR, Gabriel SE, Mengos A, Gentzsch M, Randell SH, et al. 2006. Am. J. Respir. Cell Mol. Biol. 34:355–63

127. Farinha CM, Amaral MD. 2005. Mol. Cell. Biol. 25:5242–52

128. Harada K, Okiyoneda T, Hashimoto Y, Ueno K, Nakamura K, et al. 2006. J. Biol. Chem.

281:12841–48

129. Prince LS, Workman RB Jr, Marchase RB. 1994. Proc. Natl. Acad. Sci. USA 91:5192–96

130. Picciano JA, Ameen N, Grant B, Bradbury NA. 2003. Am. J. Physiol. Cell Physiol.

285:C1009–18

131. Sharma M, Pampinella F, Nemes C, Benharouga M, So J, et al. 2004. J. Cell Biol. 164:923–

33

132. Lukacs GL, Segal G, Kartner N, Grinstein S, Zhang F. 1997. Biochem. J. 328:353–61

133. Bradbury NA, Cohn JA, Venglarik CJ, Bridges RJ. 1994. J. Biol. Chem. 269:8296–302

134. Webster P, Vanacore L, Nairn AC, Marino CR. 1994. Am. J. Physiol. 267:C340–48

135. Howard M, Jiang X, Stolz DB, Hill WG, Johnson JA, et al. 2000. Am. J. Physiol. Cell Physiol. 279:C375–82

724

Riordan

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

136. Sharma M, Benharouga M, Hu W, Lukacs GL. 2001. J. Biol. Chem. 276:8942–50

137. Lukacs GL, Chang X-B, Bear C, Kartner N, Mohamed A, et al. 1993. J. Biol. Chem.

268:21592–98

138. Swiatecka-Urban A, Brown A, Moreau-Marquis S, Renuka J, Coutermarsh B, et al. 2005.

J. Biol. Chem. 280:36762–72

139. Weixel KM, Bradbury NA. 2001. J. Biol. Chem. 276:46251–59

140. Silvis MR, Picciano JA, Bertrand C, Weixel K, Bridges RJ, Bradbury NA. 2003. J. Biol.

Chem. 278:11554–60

141. Gentzsch M, Chang XB, Cui L, Wu Y, Ozols VV, et al. 2004. Mol. Biol. Cell 15:2684–96

142. Swiatecka-Urban A, Boyd C, Coutermarsh B, Karlson KH, Barnaby R, et al. 2004. J. Biol. Chem. 279:38025–31

143. Swiatecka-Urban A, Talebian L, Kanno E, Moreau-Marquis S, Coutermarsh B, et al. 2007. J. Biol. Chem. 282:23725–36

144. Ganeshan R, Nowotarski K, Di A, Nelson DJ, Kirk KL. 2007. Biochim. Biophys. Acta

1773:192–200

145. Bates IR, Hebert B, Luo Y, Liao J, Bachir AI, et al. 2006. Biophys. J. 91:1046–58

146. Jin S, Haggie PM, Verkman AS. 2007. Biophys. J. 93:1079–88

147. Guggino WB. 2004. Proc. Am. Thorac. Soc. 1:28–32

148. Thelin WR, Chen Y, Gentzsch M, Kreda SM, Sallee JL, et al. 2007. J. Clin. Investig.

117:364–74

149. Gentzsch M, Choudhury A, Chang XB, Pagano RE, Riordan JR. 2007. J. Cell Sci.

120:447–55

150. Huang F, Kirkpatrick D, Jiang X, Gygi S, Sorkin A. 2006. Mol. Cell 21:737–48

151. Fuchs HJ, Borowitz DS, Christiansen DH, Morris EM, Nash ML, et al. 1994. N. Engl.

J. Med. 331:637–42

152. Bonsignore CL. 1998. Pediatr. Nurs. 24:258–59

153. Ostedgaard LS, Rokhlina T, Karp PH, Lashmit P, Afione S, et al. 2005. Proc. Natl. Acad. Sci. USA 102:2952–57

154. Fischer AC, Smith CI, Cebotaru L, Zhang X, Askin FB, et al. 2007. Mol. Ther. 15:756–63

155. Carroll TP, Morales MM, Fulmer SB, Allen SS, Flotte TR, et al. 1995. J. Biol. Chem.

270:11941–46

156. Limberis MP, Figueredo J, Calcedo R, Wilson JM. 2007. Mol. Ther. 9:1694–97

157. Copreni E, Penzo M, Carrabino S, Conese M. 2004. Gene Ther. 11(Suppl. 1):S67–75

158. Kerem E, Kerem B. 1996. Pediatr. Pulmonol. 22:387–95

159. Howard M, Frizzell RA, Bedwell DM. 1996. Nat. Med. 2:467–69

160. Bedwell DM, Kaenjak A, Benos DJ, Bebok Z, Bubien JK, et al. 1997. Nat. Med. 3:1280–84

161. Du M, Jones JR, Lanier J, Keeling KM, Lindsey JR, et al. 2002. J. Mol. Med. 80:595–604

162. Wilschanski M, Yahav Y, Yaacov Y, Blau H, Bentur L, et al. 2003. N. Engl. J. Med.

349:1433–41

163. Rowe SM, Rab A, Zsuzsa B, Byram K, Li Y, et al. 2006. Pediatr. Pulmonol. Suppl. 29:227

164. Clancy JP, Rowe SM, Bebok Z, Aitken ML, Gibson R, et al. 2007. Am. J. Respir. Cell Mol.

Biol. 37:57–66

165. Galietta LV, Jayaraman S, Verkman AS. 2001. Am. J. Physiol. Cell Physiol. 281:C1734–42

166. Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, et al. 2006. Am. J. Physiol. Lung Cell Mol. Physiol. 290:L1117–30

167. Verkman AS, Lukacs GL, Galietta LJ. 2006. Curr. Pharm. Des. 12:2235–47

168. Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, et al. 2002. J. Clin. Investig.

110:1651–58

www.annualreviews.org CFTR Function and Prospects for Therapy

725

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

169. Ma T, Vetrivel L, Yang H, Pedemonte N, Zegarra-Moran O, et al. 2002. J. Biol. Chem.

277:37235–41

170. Muanprasat C, Sonawane ND, Salinas D, Taddei A, Galietta LJ, Verkman AS. 2004.

J. Gen. Physiol. 124:125–37

171. Yang H, Shelat AA, Guy RK, Gopinath VS, Ma T, et al. 2003. J. Biol. Chem. 278:35079–85

172. Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, et al. 2005. Mol. Pharmacol. 67:1797–807

173. Loo TW, Bartlett MC, Wang Y, Clarke DM. 2006. Biochem. J. 395:537–42

174. Wang Y, Bartlett MC, Loo TW, Clarke DM. 2006. Mol. Pharmacol. 70:297–302

175. Carlile GW, Robert R, Zhang D, Teske KA, Luo Y, et al. 2007. ChemBioChem 8:1012–20

176. Noel S, Faveau C, Norez C, Rogier C, Mettey Y, Becq F. 2006. J. Pharmacol. Exp. Ther.

319:349–59

177. Wang Y, Loo TW, Bartlett MC, Clarke DM. 2007. Biochem. J. 406:257–63

178. Wang Y, Loo TW, Bartlett MC, Clarke DM. 2007. Mol. Pharmacol. 71:751–58

179. Moran O, Galietta LJ, Zegarra-Moran O. 2005. Cell. Mol. Life Sci. 62:446–60

180. Kalin N, Claass A, Sommer M, Puchelle E, Tummler B. 1999. J. Clin. Investig. 103:1379–

89

181. Okiyoneda T, Lukacs GL. 2007. Biochim. Biophys. Acta 1773:476–79

182. Stutts MJ, Chinet TC, Mason SJ, Fullton JM, Clarke LL, Boucher RC. 1992. Proc. Natl. Acad. Sci. USA 89:1621–25

183. Knowles MR, Clarke LL, Boucher RC. 1992. Chest 101(3 Suppl.):S60–63

184. Deterding R, Retsch-Bogart G, Milgram L, Gibson R, Daines C, et al. 2005. Pediatr. Pulmonol. 39:339–48

185. Hofmann T, Stutts MJ, Ziersch A, Ruckes C, Weber WM, et al. 1998. Am. J. Respir. Crit.

Care Med. 157:1844–49

186. Hirsh AJ, Sabater JR, Zamurs A, Smith RT, Paradiso AM, et al. 2004. J. Pharmacol. Exp. Ther. 311:929–38

187. Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, et al. 2006. N. Engl. J. Med.

354:229–40

188. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. 2006.

N. Engl. J. Med. 354:241–50

189. Callebaut I, Eudes R, Mornon JP, Lehn P. 2004. Cell. Mol. Life Sci. 61:230–42

726

Riordan

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

2008.77:701-726. of Hawaii Rev. Biochem. by University Annu.   Annual Review of Biochemistry C o n
 

Annual Review of Biochemistry

Contents

Volume 77, 2008

Prefatory Chapters

Discovery of G Protein Signaling Zvi Selinger ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 1

Moments of Discovery Paul Berg ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 14

Single-Molecule Theme

In singulo Biochemistry: When Less Is More Carlos Bustamante ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 45

Advances in Single-Molecule Fluorescence Methods for Molecular Biology Chirlmin Joo, Hamza Balci, Yuji Ishitsuka, Chittanon Buranachai, and Taekjip Ha ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 51

How RNA Unfolds and Refolds Pan T.X. Li, Jeffrey Vieregg, and Ignacio Tinoco, Jr. ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 77

Single-Molecule Studies of Protein Folding Alessandro Borgia, Philip M. Williams, and Jane Clarke ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 101

Structure and Mechanics of Membrane Proteins Andreas Engel and Hermann E. Gaub ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 127

Single-Molecule Studies of RNA Polymerase: Motoring Along Kristina M. Herbert, William J. Greenleaf, and Steven M. Block ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 149

Translation at the Single-Molecule Level R. Andrew Marshall, Colin Echeverría Aitken, Magdalena Dorywalska, and Joseph D. Puglisi ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 177

Recent Advances in Optical Tweezers Jeffrey R. Moffitt, Yann R. Chemla, Steven B. Smith, and Carlos Bustamante ♣♣♣♣♣♣ 205

Recent Advances in Biochemistry

Mechanism of Eukaryotic Homologous Recombination Joseph San Filippo, Patrick Sung, and Hannah Klein ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 229

v

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Structural and Functional Relationships of the XPF/MUS81 Family of Proteins Alberto Ciccia, Neil McDonald, and Stephen C. West ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 259

Fat and Beyond: The Diverse Biology of PPAR γ Peter Tontonoz and Bruce M. Spiegelman ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 289

Eukaryotic DNA Ligases: Structural and Functional Insights Tom Ellenberger and Alan E. Tomkinson ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 313

Structure and Energetics of the Hydrogen-Bonded Backbone in Protein Folding D. Wayne Bolen and George D. Rose ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 339

Macromolecular Modeling with Rosetta Rhiju Das and David Baker ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 363

Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry Benjamin F. Cravatt, Aaron T. Wright, and John W. Kozarich ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 383

Analyzing Protein Interaction Networks Using Structural Information Christina Kiel, Pedro Beltrao, and Luis Serrano ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 415

Integrating Diverse Data for Structure Determination of Macromolecular Assemblies Frank Alber, Friedrich Förster, Dmitry Korkin, Maya Topf, and Andrej Sali ♣♣♣♣♣♣♣♣ 443

From the Determination of Complex Reaction Mechanisms to Systems Biology John Ross ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 479

Biochemistry and Physiology of Mammalian Secreted Phospholipases A 2 G´erard Lambeau and Michael H. Gelb ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 495

Glycosyltransferases: Structures, Functions, and Mechanisms L.L. Lairson, B. Henrissat, G.J. Davies, and S.G. Withers ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 521

Structural Biology of the Tumor Suppressor p53 Andreas C. Joerger and Alan R. Fersht ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 557

Toward a Biomechanical Understanding of Whole Bacterial Cells Dylan M. Morris and Grant J. Jensen ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 583

How Does Synaptotagmin Trigger Neurotransmitter Release? Edwin R. Chapman ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 615

Protein Translocation Across the Bacterial Cytoplasmic Membrane Arnold J.M. Driessen and Nico Nouwen ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 643

vi

Contents

www.annualreviews.org

personal use only.

from For

on 01/25/11.

Downloaded

at Manoa Library

2008.77:701-726.

of Hawaii

Rev. Biochem.

by University

Annu.

Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms, Connected Processes, and Diseases Roland Lill and Ulrich Mühlenhoff ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 669

CFTR Function and Prospects for Therapy John R. Riordan ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 701

Aging and Survival: The Genetics of Life Span Extension by Dietary Restriction William Mair and Andrew Dillin ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 727

Cellular Defenses against Superoxide and Hydrogen Peroxide James A. Imlay ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 755

Toward a Control Theory Analysis of Aging Michael P. Murphy and Linda Partridge ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 777

Indexes

Cumulative Index of Contributing Authors, Volumes 73–77 ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 799

Cumulative Index of Chapter Titles, Volumes 73–77 ♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣♣ 803

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found at http://biochem.annualreviews.org/errata.shtml