Structure and Regulation of Soluble Guanylate Cyclasematzman/Files/FCT/sGC14/7_Derbyshire_2012.pdf · sine monophosphate (cGMP) pathway as it functions as the primary activator of

BI81CH22-Marletta ARI 3 May 2012 12:17

Structure and Regulation ofSoluble Guanylate CyclaseEmily R. Derbyshire1 and Michael A. Marletta2

1Department of Biological Chemistry and Molecular Pharmacology, Harvard MedicalSchool, Boston, Massachusetts 021152Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037;email: [email protected]

Annu. Rev. Biochem. 2012. 81:533–59

First published online as a Review in Advance onFebruary 9, 2012

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev-biochem-050410-100030

Copyright c© 2012 by Annual Reviews.All rights reserved

0066-4154/12/0707-0533$20.00

Keywords

nitric oxide, heme, signaling, desensitization, nitrosation

Abstract

Nitric oxide (NO) is an essential signaling molecule in biological sys-tems. In mammals, the diatomic gas is critical to the cyclic guano-sine monophosphate (cGMP) pathway as it functions as the primaryactivator of soluble guanylate cyclase (sGC). NO is synthesized fromL-arginine and oxygen (O2) by the enzyme nitric oxide synthase (NOS).Once produced, NO rapidly diffuses across cell membranes and bindsto the heme cofactor of sGC. sGC forms a stable complex with NOand carbon monoxide (CO), but not with O2. The binding of NO tosGC leads to significant increases in cGMP levels. The second mes-senger then directly modulates phosphodiesterases (PDEs), ion-gatedchannels, or cGMP-dependent protein kinases to regulate physiologicalfunctions, including vasodilation, platelet aggregation, and neurotrans-mission. Many studies are focused on elucidating the molecular mech-anism of sGC activation and deactivation with a goal of therapeuticintervention in diseases involving the NO/cGMP-signaling pathway.This review summarizes the current understanding of sGC structureand regulation as well as recent developments in NO signaling.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 534Enzymes Critical to the Nitric

Oxide/Cyclic GuanosineMonophosphate Pathway. . . . . . . . 534

ISOLATION OF SOLUBLEGUANYLATE CYCLASE . . . . . . . . 535

SOLUBLE GUANYLATECYCLASE ISOFORMS . . . . . . . . . . . 537

ARCHITECTURE OF SOLUBLEGUANYLATE CYCLASE . . . . . . . . 538Heme-Nitric Oxide and Oxygen

Binding Domain . . . . . . . . . . . . . . . . 538Per/Arnt/Sim and Coiled-Coil

Domains . . . . . . . . . . . . . . . . . . . . . . . 539Catalytic Domain . . . . . . . . . . . . . . . . . . 539

SOLUBLE GUANYLATECYCLASE HOMOLOGS . . . . . . . . . 540Eukaryotic Atypical Soluble

Guanylate Cyclases . . . . . . . . . . . . . 540Prokaryotic Heme-Nitric Oxide

and Oxygen BindingProteins . . . . . . . . . . . . . . . . . . . . . . . . 541

STRUCTURAL INSIGHTS FROMSTUDIES ON SOLUBLEGUANYLATE CYCLASE ANDITS HOMOLOGS . . . . . . . . . . . . . . . . 542

LIGAND SELECTIVITY . . . . . . . . . . . . 545REGULATION OF SOLUBLE

GUANYLATE CYCLASE BYNITRIC OXIDE . . . . . . . . . . . . . . . . . . 546Activation and Nitric Oxide

Association . . . . . . . . . . . . . . . . . . . . . 546Deactivation and Nitric Oxide

Dissociation . . . . . . . . . . . . . . . . . . . . 547Desensitization . . . . . . . . . . . . . . . . . . . . 548

MODULATORS OF SOLUBLEGUANYLATE CYCLASEACTIVITY . . . . . . . . . . . . . . . . . . . . . . . 549Soluble Guanylate Cyclase

Activators . . . . . . . . . . . . . . . . . . . . . . 549Soluble Guanylate Cyclase

Inhibitors. . . . . . . . . . . . . . . . . . . . . . . 550

INTRODUCTION

The nitric oxide/cyclic guanosine monophos-phate (NO/cGMP) pathway was discoveredin the 1980s, but chemical modulation of thepathway for the treatment of angina pectorishad been unknowingly achieved 100 yearsearlier. This stimulation of cGMP productionoccurred with the clinical administration oforganic nitrites (isoamyl nitrite) (1) and organicnitrates (glyceryl trinitrate; GTN) (2). Thesecompounds alleviate the pain associated withangina by relaxing the vascular smooth muscle,leading to vasodilation. For years, investiga-tions were focused on the mechanism of smoothmuscle relaxation by these molecules, and theseefforts led to the discovery that NO is a physio-logically relevant signaling molecule. Addition-ally, these efforts led to the identification of theenzymes that biosynthesize NO and cGMP.

Enzymes Critical to the NitricOxide/Cyclic GuanosineMonophosphate Pathway

Early studies showed that both cytosolic andparticulate fractions of mammalian tissueexhibit guanylate cyclase activity. Withinthe insoluble fractions, membrane-boundparticulate guanylate cyclases are present,which are activated by natriuretic peptides(reviewed in References 3 and 4), whereas thecytosolic fractions contain soluble guanylatecyclases (sGCs), which are activated by NO.NO-responsive guanylate cyclase activity isalso associated with cell membranes in certaintissues, including skeletal muscle and brain, aswell as in platelets (5–7). Guanylate cyclasesare found in most tissues, and the distributionof these proteins in various cells is isoformspecific. This provides an additional means toregulate cGMP-dependent responses becauselocalized pools of the signaling molecule canbe generated within specific tissues and inproximity to either soluble or membrane-bound cGMP receptors. Thus, tissues can

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cGMP: cyclicguanosinemonophosphate

GTN: glyceryltrinitrate

Vasodilation: bloodvessel widening fromsmooth musclerelaxation

sGC: solubleguanylate cyclase

NOS: nitric oxidesynthase

regulate cGMP levels by expression of specificGC isoforms, and the isoforms have a distinctpeptide receptor or ligand activator. Addi-tionally, a reciprocal communication betweenparticulate guanylate cyclase and sGC has beenobserved in the regulation of human and mousevascular homeostasis (8), and it remains likelythat communication between these pathwaysoccurs in several cGMP-dependent processes.

Generally, in eukaryotic NO signaling,the initial event involves calcium release,followed by binding of a calcium/calmodulincomplex to nitric oxide synthase (NOS), whichactivates the enzyme. NO is synthesized andthen diffuses into target cells, where it bindsto the heme in sGC (Figure 1). sGC is ahistidine-ligated hemoprotein that binds NOand carbon monoxide (CO), but not oxygen(O2). This binding event leads to a severalhundredfold increase in cGMP synthe-sis. Once formed, cGMP targets include

phosphodiesterases (PDEs), ion-gated chan-nels, and cGMP-dependent protein kinasesin the regulation of several physiologicalfunctions, including vasodilation, plateletaggregation, and neurotransmission (9–11).

In 1998, the Nobel Prize in Physiology orMedicine was awarded to Robert F. Furchgott,Louis J. Ignarro, and Ferid Murad, in recogni-tion of their achievements toward the discoveryof the NO-signaling pathway. Currently, thispathway is actively studied because drugs mod-ulating NO-dependent processes have the po-tential to treat several maladies, including car-diovascular and neurodegenerative diseases, aswell as various airway diseases.

ISOLATION OF SOLUBLEGUANYLATE CYCLASE

Despite many years of research on sGC, an ef-ficient low-cost purification of the protein has

Generator cell Target cell

NOS sGC

GTP

FeII

α1 β1

NO

cGKPDE

cGMP-gatedion channels

Ca2+/CaM

L-Arg + O2

L-Cit+

NO

Mg2+

cGMP + PPi

cell Ta

α1 β1

Figure 1The nitric oxide/cyclic GMP (NO/cGMP)-signaling pathway. A Ca2+/calmodulin (CaM) complex bindsnitric oxide synthase (NOS). NOS catalyzes the oxidation of L-arginine (L-Arg) to L-citrulline (L-Cit) andnitric oxide (NO). NO binds to the FeII heme of α1β1 soluble guanylate cyclase (sGC) at a diffusion-controlled rate. This binding event leads to significant increases in cGMP and pyrophosphate (PPi). cGMPthen binds to and activates cGMP-dependent protein kinases (cGKs), phosphodiesterases (PDEs) andion-gated channels. Abbreviations: α1 and β1, soluble guanylate cyclase subunits; CaM, calmodulin.

www.annualreviews.org • Structure and Regulation of SGC 535

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GTP: guanosine5′-triphosphate

remained elusive, but several methods havebeen developed that yield low microgramamounts of homogeneous protein. Initial char-acterization of sGC was carried out with pro-tein obtained from rat and bovine tissues. Bythe 1980s, studies were being done with pu-rified sGC from rat lung (12) and liver (13),as well as from bovine lung (14, 15); thesestudies showed sGC to be a heterodimer. Im-portantly, it was observed that sGC could bepurified with and without the heme cofac-tor, depending on the purification protocol.In short, the use of solubilizing agents or am-monium sulfate precipitation can lead to mis-folded apoprotein. Heme reconstitution of thisapoprotein yields an sGC species [later termedsGC1 by Vogel et al. (16)] with biochemi-cal properties that vary from the native pro-tein [named sGC2 by Vogel et al. (16)]. Todate, the bovine lung sGC prep is the most ef-ficient method of isolating heme-bound pro-tein from source tissue (17, 18). This methodtypically yields ∼1 mg of pure protein perkilogram of lung.

The development of heterologous expres-sion systems for recombinant sGC expressionled to significant advances. The first successfulheterologous expression system for sGC wasaccomplished in COS-7 cells (19). AlthoughCOS-7 cells do not produce enough sGC forprotein purification, the procedure was pivotalto establishing that sGC is an obligate het-erodimer composed of α1 and β1 subunits (20).Additionally, COS-7 cells have been used toexamine mutants and truncations of sGC vialysate activity assays (21, 22).

The overexpression of rat sGC in insect cellswith the Sf9/baculovirus expression system wasthe first procedure used to isolate pure recom-binant protein (21, 23, 24). sGC expression ininsect cells was initially accomplished withoutan affinity tag (21), but current protocolsinvolve a His tag to facilitate the purificationprocess (25–27). Although highly expressed,most (>90%) of the protein is insoluble. Thismethod typically yields 0.2–0.4 mg of pure sol-uble protein per liter of culture and is now com-monly used to obtain purified rat and human

sGC. A clear advantage of the Sf9/baculovirusexpression system is that it enables in vitrocharacterization, including the generationof site-directed mutants. However, both theCOS-7 and Sf9/baculovirus expression systemsfacilitated the biochemical characterization ofsubunit dimerization, allowed for the gener-ation of site-directed mutants, and providedlarger quantities of enzyme for in vitro studies.

The full-length mammalian α1β1 het-erodimer has not yet been isolated from a bac-terial expression system, but several truncationsof sGC have been successfully obtained via ex-pression in Escherichia coli. These proteins in-clude N-terminal truncations of α1, β1, andβ2 (28–30), C-terminal truncations of α1 andβ1 (31, 32), and a domain within the central re-gion of β1 (Figure 2) (31). These constructspurify with yields ranging from 0.5 to 5 mg

b Characterized sGC truncations

a Domain architecture of sGC

β1(1–385)

α1(467–690)

β1(414–619)

β1(1–194)

β2(1–217)

Heme binding

GTP binding

H-NOX PAS CC CAT

α

β

Heme

Figure 2Domain architecture of soluble guanylate cyclase(sGC). (a) Heme-nitric oxide/oxygen binding(H-NOX, yellow), Per/Arnt/Sim domain (PAS,gray), coiled-coil domains (CC, white), and catalyticdomains (CAT, blue) are shown. (b) Characterizedheme-binding and GTP-binding truncations of ratsGC. Heme is represented by the red parallelogram.α1, β1, and β2 isoforms are shown.


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of pure protein per liter of culture. Recentlyan E. coli expression system was used for thefull-length Manduca sexta α1β1 heterodimer(33). This method provides low amounts (0.5–1 mg/liter) of partially pure full-length protein,but higher yields (1–2 mg/liter) of pure proteinwere obtained by truncating the C terminus ofthe α1 and β1 subunits (33). The resulting het-erodimeric proteins (msGC-NT1 and msGC-NT2) lack the ability to cyclize GTP but can bepurified to homogeneity. Perhaps future workoptimizing the expression conditions and/orpurification of sGC from E. coli will overcomethe current limitations in protein yield.

SOLUBLE GUANYLATECYCLASE ISOFORMS

Heterodimeric sGC consists of two homolo-gous subunits, α and β. The most commonlystudied isoform is the α1β1 protein; however,α2 and β2 subunits have also been identified(34, 35). These proteins were first character-ized in mammals, but they also exist in insects,such as Drosophila melanogaster and M. sexta, andin fish. Isoforms of α-subunits are highly ho-mologous with ∼48% sequence identity, andβ-subunits have an overall sequence identity of∼41%.

The localization of each subunit has beenstudied in mammals, including humans, rats,and cows. Both α1 and β1 subunits are ex-pressed in most tissues, and it is well acceptedthat these proteins form a physiologicallyrelevant heterodimer (36). By quantitativepolymerase chain reaction analysis and West-ern blotting, the α2 subunit is found in fewertissues when compared to the α1 and β1 iso-forms but is highly expressed in the brain, lung,colon, heart, spleen, uterus, and placenta (36).Studies with purified protein have shown thatthe α2β1 heterodimer exhibits ligand-bindingcharacteristics identical to the α1β1 het-erodimer (37, 38), but a splice variant of the α2subunit (α2i) forms a dimer with the β1 subunitto form an inactive complex. α2i contains anin-frame insertion of 31 amino acids withinthe catalytic domain and appears to function as

a dominant-negative protein (39). Despite thesimilar biochemical properties of the two phys-iologically relevant sGC heterodimers, theyhave unique roles in cGMP signaling that maybe attributed to their varying cellular local-ization. Specifically, α2β1 has been associatedwith the membrane in several tissues (5–7),and consequently, α2β1 responds differentlythan cytosolic α1β1 (37). In rat brain, it wasfound that this association is mediated by aninteraction between the C terminus of the α2protein and PSD-95 (postsynaptic density-95)protein (6). Most recently, a distinct presy-naptic role has been identified for α1β1 inglutamate release in the hippocampus (40).

The β2 isoform is not ubiquitously ex-pressed like the β1 isoform, and analysis ofmRNA levels indicates that it is found primarilyin the kidney (35). Unlike β1, the C terminusof β2 contains a possible isoprenylation site,but the subcellular localization of this proteinis unknown. The β2 isoform has not yet beenpurified and characterized, but the protein hasbeen studied after transient expression in insectcells. Using this approach, it was found that β2does not exhibit cyclase activity when expressedwith α1 or α2 but that β2 is active in the absenceof an α-subunit, suggesting that the β2 proteincan function as a homodimer ex vivo (41). Thisis in contrast to the β1 protein, which has beenisolated as an inactive homodimer after overex-pression in insect cells (26). In rat kidney, themRNA levels of the β2 subunit were shown tobe developmentally regulated (42); however, itremains unclear what the physiological role ofthe β2 isoform is in cGMP signaling.

Recently, several genetic studies in mousemodels have emphasized the importance ofthe various sGC isoforms for physiologicalprocesses (reviewed in Reference 43). Knock-out mice lacking the sGC β1 subunit exhibitelevated blood pressure, reduced heart rate,and dysfunction in gastrointestinal contractility(44). Additionally, deletion of the β1 subunitwithin only smooth muscle cells implicates theloss of the protein in these cells as the causeof hypertension in the knockout mice (45).Deletion of the β1 subunit is generally viewed


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PAS: protein foldnamed for itsassociation with thePer, ARNT, and Simproteins

H-NOX: heme-nitricoxide and oxygenbinding

as a global sGC knockout because α1 and α2do not form functional heterodimers with β2.sGC α1 and α2 knockout mice have also beengenerated. Both proteins were found to beessential for long-term potentiation (46), andvasodilation was mediated primarily by the α1isoform (47). In addition to clarifying the role ofthe α1β1 protein in NO-mediated pulmonaryvasodilation (48, 49), studies with α1 subunit-deficient mice suggest that both α-subunits areinvolved in gastric nitrergic relaxation (50) andrelaxation of colon tissue (51).

Several invertebrates also contain genesthat encode predicted NO-sensitive guanylatecyclases. Likely owing to the genetic toolsavailable in D. melanogaster, this organismcontains the best-characterized insect guany-late cyclase. In D. melanogaster, Gycα-99Band Gycβ-100B are orthologs of the α1 andβ1 subunits, respectively. Drosophila mutantsdeficient in the production of these proteinssuggest that the NO/cGMP pathway mediatesa behavioral phenotype (52) and developmentof the visual system (53), and this pathway isimportant for larval foraging locomotion (54).In other organisms, several biochemical studieshave been aimed at elucidating the significanceof the NO/cGMP-signaling pathway. As men-tioned above, the full-length M. sexta α1β1heterodimer has recently been characterized(33). There is evidence that sGC in M. sexta isinvolved in neuronal excitability (55) and odorresponsiveness (56). In mollusks, such as theLimax marginatus and Limax maximus slugs,sGC may modulate the electrical oscillation ofinterneurons in the central olfactory pathway(57, 58).

ARCHITECTURE OF SOLUBLEGUANYLATE CYCLASE

The rat sGC α1 and β1 subunits are 690and 619 amino acids in length, respectively.These proteins are part of a large family of sGCsubunits that are conserved in eukaryotes. Gen-erally, there is the highest sequence variabilityat the N terminus of α-subunits and the great-est sequence identity at the C terminus of both

the α- and β-proteins. Each sGC subunit con-sists of four distinct domains. The β1 subunitcontains a N-terminal heme-binding domain,a Per/Arnt/Sim (PAS) domain, a coiled-coildomain, and a C-terminal catalytic domain(Figure 2) (reviewed in Reference 59).

Heme-Nitric Oxide and OxygenBinding Domain

Experiments with sGC truncations and site-directed mutants were necessary to localize theminimal heme-binding domain of sGC. Theseexperiments involved the systematic mutationof conserved histidines (60), expression of var-ious truncations in E. coli (29), and deletionof the β1 N terminus (61). Taken together,these studies showed that the β1 N terminusconstituted the heme-binding domain and sug-gested that histidine 105 (rat) was the proximalheme ligand. The sGC heme-binding domainhas been localized to residues 1 to ∼194 onthe β1 subunit (28). Like the full-length sGC,the isolated heme domain binds NO and CO,but not O2. The N terminus of the α1 subunithas homology to the β1 N terminus and wasshown to have affinity for heme despite lackingthe proximal histidine ligand (30). The sGC Nterminus is part of a conserved family of pro-teins found in both prokaryotes and eukaryotes(62). This family of proteins has been termedheme-nitric oxide binding (62), sensor of nitricoxide (63), and heme-nitric oxide and oxygenbinding (H-NOX) (64). H-NOX is the mostused abbreviation and will be used through-out this review. To date, all of the character-ized H-NOX proteins bind heme as well as thegaseous heme ligands NO and CO (reviewed inReference 65). Some H-NOX proteins, includ-ing β1 and β2, discriminate against O2 bind-ing, whereas others form a stable complex withO2. In eukaryotes, H-NOX proteins have onlybeen found with the known sGC domain ar-chitecture. In bacteria, H-NOX domains canbe found as proteins of ∼200 amino acids inlength with a single predicted function or as adomain within a larger protein on the basis ofsequence analysis programs. Additionally, the


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genes that encode these H-NOX proteins inbacteria are in proximity to genes that encodeputative histidine kinases and diguanylate cy-clases, or in some cases, a gene encodes theH-NOX as a domain within a larger protein,often fused to a methyl-accepting chemotaxisdomain. It is likely that these proteins have anevolutionarily conserved function and serve asgas sensors in prokaryotes and eukaryotes.

Per/Arnt/Sim and Coiled-CoilDomains

The central region of sGC contains twodomains of unresolved function. The domaincloser to the N terminus is predicted to adopt aPAS-like fold. Typically, PAS domains mediateprotein-protein interactions and have oftenbeen found to bind heme, a flavin, or a nu-cleotide (66). The other domain, a coiled-coildomain, appears to be unique to sGC and sharesno significant homology with any other proteinin the National Center for Biotechnology In-formation protein database (http://www.ncbi.nlm.nih.gov/protein). The coiled-coil do-main of the rat β1 subunit (residues 348–409)was isolated as a tetramer and structurallyelucidated with X-ray crystallography (31).This structural study, in addition to experi-ments involving site-directed mutagenesis ofresidues on the α1 subunit (67), a bimolecularfluorescence complementation assay in cells(68), and structural analysis of homologs of thesGC PAS domain (69), suggests that the centralregions of both sGC subunits are importantfor the formation of a functional heterodimer.

Catalytic Domain

The C-terminal regions of the α1 and β1 pro-teins are highly homologous to the particulateguanylate cyclase and adenylate cyclase catalyticdomains. In the 1990s, structural insights onthe sGC catalytic domains came from homol-ogy models on the basis of crystal structures ofthe adenylate cyclase catalytic domains (70, 71).These models identified key catalytic residues,including two conserved aspartate residues on

the α1 subunit (D485 and D529, rat number-ing), which are predicted to bind two Mg2+

ions (72). These residues are critical to catal-ysis as the associated metals likely activate boththe nucleotide 3′-hydroxyl and the α-phosphatefor the reaction, as well as stabilize the chargeon the β- and γ-phosphates on both the sub-strate and product. Additionally, β1 N548 isproposed to orient the ribose ring for the re-action. Residues thought to be responsible forbase recognition include E473 and C541 on theβ1 subunit. Other residues on both α1 (R573)and β1 (R552) are thought to interact with thenucleotide triphosphate (72). With the identi-fication of these critical residues, predictionscan be made about guanylate cyclase activityusing sequence analysis. This type of analysiswould correctly predict that the β2 isoformcould function as a homodimer but that β1, α1,and α2 need a partner to be active.

The catalytic domains have now been local-ized to the C-terminal 467–690 and 414–619residues of the α1 and β1 subunits, respectively(32). These catalytic domains must form aheterodimer for cGMP to be synthesized, andin the full-length protein, the catalytic effi-ciency of the protein is dependent on the hemeligation state of the β1 H-NOX domain. sGCis highly selective for GTP as a substrate, butthe protein can also cyclize 2′-d-GTP, GTP-γ-S, guanosine 5′-[β,γ-imido]-triphosphate(GMP-PNP), ITP, UTP, and ATP (73–75).The isolated α1catβ1cat heterodimer is alsoselective for GTP but can synthesize cAMPfrom ATP (76). Interestingly, the activity ofα1catβ1cat is inhibited by the presence of theH-NOX domain [β1(1–194) or β1(1–385)](32).1,2 This shows that these domains interactin trans and suggests that the NO mechanismof activation involves the relief of an inhibitoryinteraction between the H-NOX domainand the catalytic domains. In support of this

1α1cat and β1cat are the catalytic domains of the rat sGC α1and sGC β1 subunits, respectively.2β1(1–194) represents residues 1–194 of the rat sGC β1subunit, the minimum heme-binding domain.


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http://www.ncbi.nlm.nih.gov/protein

http://www.ncbi.nlm.nih.gov/protein


sGC homologs:proteins with highsequence homology tomammalian sGCs

Atypical sGCs:distinct subset of sGCswith reducedsensitivity to NO

proposal, a fluorescence (or Forster) resonanceenergy transfer-based study showed that theN terminus of both the α1 and α2 subunitsinteract with the C terminus of the β1 subunit(77). In addition to the heterodimeric rat sGCcatalytic domains, the catalytic domain fromthe Chlamydomonas reinhardtii sGC (CYG12)has been biochemically characterized (78).This protein, as well as Synechocystis PCC6803Cya2, a particulate guanylate cyclase (79), isdiscussed in more detail below.

SOLUBLE GUANYLATECYCLASE HOMOLOGS

After the initial identification in mammals, itwas not until the emergence of genome se-quencing that the prevalence of sGC and sGChomologs in other organisms was fully realized.Full-length guanylate cyclases with domain ar-chitecture similar to the α1 and β1 subunitswere found in several eukaryotic organisms. Inprokaryotes, sGC-like H-NOX domains werefound as stand-alone proteins or fused to otherfunctional domains (62). In some genomes, ansGC-like PAS domain was also identified. Todate, the genes for sGC-like PAS domains arealways found near genes that encode sGC-likeH-NOX domains (62).

Eukaryotic Atypical SolubleGuanylate Cyclases

As mentioned above, an increasing numberof eukaryotic organisms are known to containpredicted sGCs. Some sGCs are very similarto the well-characterized rat α1 and β1 sub-units, whereas others vary significantly. Collec-tively, these cyclases have been termed atypicalsGCs (80), owing to their distinct activation anddimerization properties. Some atypical sGCsexhibit a very surprising property—the abilityto respond to O2.

The D. melanogaster genome contains fivegenes that code for sGCs. Two of these genescode for subunits with high homology to the α1and β1 proteins and have been shown to forma highly NO-sensitive heterodimeric sGC(Gycα-99B and Gycβ-100B). The other three

genes code for subunits with greater homologyto the β2 protein (Gyc-88E, Gyc-89Da,and Gyc-89Db) (81). Like β2, Gyc-88E canfunction as a homodimer, whereas Gyc-89Daand Gyc-89Db are only active as heterodimers(81). Experiments with cells overexpressingGyc-88E, Gyc-89Da, and Gyc-89Db indicatethat cGMP synthesis in these cyclases is acti-vated in the absence of O2 (80), and work withpurified protein confirms that the Gyc-88Ehomodimer forms a stable complex with O2

(82). As expected, Gyc-88E also binds NOand CO, but there are data that support therole of these proteins in mediating behav-ioral responses in hyperoxic environments inD. melanogaster (83). Interestingly, Gyc-88Eis inhibited two- to threefold by the bindingof NO, CO, and O2, a property that is quitedistinct from the ligand-induced activation ofthe sGC α1β1 heterodimer.

On the basis of sequence analysis, atypi-cal sGCs exist in several organisms, includ-ing Caenorhabditis elegans (GCY-31-GCY-37);however, worms do not contain a predictedNOS or NO-sensitive sGC. Seven β-likeguanylate cyclases are contained within theC. elegans genome, and it is likely that each genehas an important functional role. GCY-35 is in-volved in social feeding (84), and both GCY-35and GCY-36 promote aggregation and border-ing behaviors (85). Significantly, these cGMP-dependent behavioral responses are mediatedby O2, which suggests the proteins function asO2 sensors in vivo (84, 86). In support of thisproposal, the GCY-35 H-NOX domain wasisolated and shown to bind O2 (84). Analysis ofgcy-31 and gcy-33 mutants also implicates thesegenes in O2-dependent behavioral responses inC. elegans, and the proteins encoded by thesegenes (GCY-31 and GCY-33) likely functionin distinct sensory neurons (BAG versus URX)when compared to GCY-35 and GCY-36 (87).

Thus, a distinct class of cyclases exist, whichbind O2 and are inhibited by ligand binding, butthere is currently no means to predict if a cy-clase is activated or inhibited by gaseous ligandbinding on the basis of sequence analysis. How-ever, residues that contribute to gaseous ligand


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selectivity have been identified, as describedbelow.

Prokaryotic Heme-Nitric Oxideand Oxygen Binding Proteins

Several species of bacteria are known to containH-NOX proteins, and this number continuesto increase as more genomes are sequenced. In-terestingly, all of the isolated H-NOX domainsfrom facultative aerobes have ligand-bindingproperties like sGC, namely they do notbind O2. In facultative aerobes, the bacterialmembers of the H-NOX family encode a singledomain as a predicted stand-alone protein,and genes that encode either putative histidinekinases or diguanylate cyclases are found inthe same predicted operon, suggesting that thedomain has a role in two-component signalingin bacteria. In support of this hypothesis, anH-NOX domain and a predicted histidinekinase from Shewanella oneidensis were isolatedand found to interact in vitro. Additionally, thefunctional interaction between the H-NOXand kinase was mediated by NO (88). Vibrio fis-cheri also contains an H-NOX protein, termedH-NOXVf , which is proposed to regulate a pu-tative histidine kinase. In V. fischeri, H-NOXVf

was shown to regulate genes involved in ironuptake, and these same genes are modulatedby the presence of NO (89), suggesting thatH-NOXVf mediates gene expression by sensingNO. V. fischeri colonizes the light-emittingorgan of the Hawaiian bobtail squid, Euprymnascolopes, and it is likely that this mutualistichost-microbe symbiosis is mediated by aNO/H-NOXVf interaction. Some bacteria,like Rhodobacter sphaeroides and Nostoc puncti-forme, also encode a predicted PAS-like domainupstream of the H-NOX gene (62). The func-tional significance of this PAS domain tobacterial signaling is unknown, but it has beenproposed to mediate protein dimerization (69).

In obligate anaerobes, such as Thermo-nanaerobacter tengcongensis, the N-terminalH-NOX domain is fused to a C-terminalmethyl-accepting chemotaxis protein, suggest-ing a role in a chemotactic/signaling function.This H-NOX domain was found to form a

NITRIC OXIDE SIGNALING IN BACTERIA

A currently expanding topic within biological studies on NO in-volves the role of the diatomic gas in bacterial signaling. NOS-likeproteins have been identified in several prokaryotic organisms,including Bacillus anthracis, Bacillus subtilis, Sorangium cellulosum,and Streptomyces turgidiscabies. Additionally, a wide range of bac-teria also have nitrite reductases, which generate NO as part ofdenitrifying, assimilatory, and dissimilatory pathways. This en-dogenously produced NO is known to regulate several transcrip-tion factors via S-nitrosation. There are also two potential classesof prokaryotic heme-based NO sensors: globin-like proteins andH-NOX proteins. Microbial globin-like proteins bind O2, NO,and CO, and are thought to be involved in the nitrosative stressresponse. Some H-NOX proteins bind O2, in addition to COand NO, whereas others exclude O2 binding. The genes thatcode for these H-NOX proteins are found in the same operonsas predicted histidine kinases or diguanylate cyclases. In vitrostudies have shown that the H-NOX protein and histidine kinasefrom S. oneidensis interact in a NO-dependent manner, but thephysiological significance of this interaction is unknown. How-ever, NO is known to be important for the symbiosis between thebobtail squid and V. fischeri, where the H-NOX from V. fischeriregulates colonization of the bacteria within the symbiotic hostsquid by a mechanism that is dependent on NO. Further experi-ments in different microbial systems will likely uncover additionalNO-dependent signaling processes in bacteria.

very stable heme-O2 complex (Kd = 90 nM)(90), a molecular distinction from the H-NOXdomains from aerobic bacteria. The isolationand characterization of the T. tengcongensisH-NOX domain have significantly influencedcurrent understanding of sGC because it wasthe first H-NOX domain to be structurallydetermined, and, moreover, it was crystallizedbound to the diatomic ligand O2 (63, 64).

Thus far, these proteins have been used astools for probing sGC structure and regula-tion, but functional studies in microbial systems(see the sidebar titled Nitric Oxide Signalingin Bacteria) will be particularly interesting asthe variable ligand-binding properties of theseH-NOXs may have consequences for their abil-ity to respond to different gases, i.e., some pro-teins may sense O2 in addition to NO or CO.


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H-NOX domain PAS domain Catalytic domain CC domain

Figure 3Structures of heme nitric oxide and oxygen binding (H-NOX), PAS, coiled-coil (CC), and catalytic domains. The structures shown arethe T. tengcongensis H-NOX domain [Protein Data Bank (pdb) code 1U55], the dimerized PAS domain from N. punctiforme PCC 73102(pdb 2p04), the CC domain from the rat β1 subunit (pdb 3hls), and the homodimeric guanylate cyclase domain from C. reinhardtii(pdb 3et6).

YC-1:3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole

STRUCTURAL INSIGHTSFROM STUDIES ON SOLUBLEGUANYLATE CYCLASE ANDITS HOMOLOGSAn increasing number of sGC homologs havebeen isolated and characterized. Significantly,some of these homologs have been amenableto crystallography, thus providing a foundationfor structural proposals on the β1 H-NOX,PAS, and catalytic domains. The first crystalstructure of an sGC-like domain was that ofthe O2-binding H-NOX domain (residues1–188) from T. tengcongensis (Figure 3) (63,64). This protein crystallized in two differentsix-coordinate heme states, with O2 boundto the reduced heme iron and in the oxi-dized heme state. These reports identifiedseveral amino acids with critical structuralroles that are highly conserved within theH-NOX family. Among the highly conservedamino acids was the T. tengcongensis H-NOXheme-coordinating histidine (H102) andthree residues that stabilize heme binding(Figure 4). Specifically, arginine (R135) is crit-ical to the coordination of the heme propionategroups and forms a hydrogen bond with bothcarboxyl groups, and tyrosine (Y135) and serine(S133) coordinate to one of the heme carboxylgroups. Together, these residues form a YxSxRmotif that is strictly conserved in H-NOX

proteins. The proximal histidine, arginine,and tyrosine had been previously identified ascritical residues for heme binding in the sGC β1subunit on the basis of mutagenesis studies (91,92), and the role of these residues became clearwhen the T. tengcongensis H-NOX structure wassolved.

Another striking characteristic of theT. tengcongensis H-NOX structure is a highlynonplanar heme conformation; it contains oneof the most highly distorted hemes reported inthe Protein Data Bank. Interestingly, differentmolecules of the O2-bound H-NOX structureexhibit varying degrees of deformation, sug-gesting that the heme can exist in a range of con-formations. This proposal is supported by theobservation that the crystal structure of the H-NOX protein from Nostoc sp. contains a moder-ately distorted heme (93). This heme distortionis not an artifact of crystallization as it is alsoobserved in solution on the basis of an NMRstudy of the S. oneidensis H-NOX (94) and reso-nance Raman experiments with T. tengcongensisH-NOX (95) and α1β1 sGC (96–98). In fact,the dynamic range of heme conformations canbe accessed in solution by site-directed muta-genesis (95) or, in the case of sGC, by additionof the allosteric activators YC-1 or BAY 41-2272 (96–98). Specifically, the α1β1 sGC hemebecomes more planar upon activator binding;


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αG

αF

αA

αD

Y140

H102

W9

N74

Figure 4Distal heme pocket of the T. tengcongensis H-NOX domain [Protein Data Bank (pdb) 1U55 code]. Residuesimportant to ligand selectivity (W9, N74, and Y140) and heme binding (H102) are shown. Imagereproduced with permission of Elsevier (65).

however, it is unclear if this is a cause or con-sequence of enzyme activation. Thus, althoughthe functional importance of heme distortionin sGC activation remains unknown, there isa potential to utilize changes in distortion inbiological responses.

Among the residues thought to be critical tomaintaining the nonplanar heme conformationare I5, D45, R135 (in the YxSxR motif ), L144,and P115, all of which are highly conservedin H-NOX proteins (64). In T. tengcongensisH-NOX, mutation of proline 115 to alanineleads to relaxation of the distorted heme (95,99). This heme relaxation was not observed af-ter mutation of proline 118 to alanine (P115 inT. tengcongensis H-NOX) in α1β1 sGC (96);however, it is not surprising that additionalresidues or domain interactions are importantfor maintaining the heme conformation in themammalian protein.

The crystal structure of the non-O2-bindingH-NOX from Nostoc sp. has been solved inthe five-coordinate unligated state and as thesix-coordinate NO- and CO-heme complexes(93). Nostoc sp. H-NOX could function as aredox or NO sensor (100). Comparison of theT. tengcongensis H-NOX and Nostoc sp. H-NOXstructures in different ligation states (FeII-CO,FeII-NO, and FeII-unligated states) led to

speculation on a molecular mechanism of sGCactivation. Specifically, the differential pivotingand bending in the H-NOX heme upon NO orCO binding was suggested to account for thevarying degree of activation induced by the twoligands (200-fold versus fourfold, respectively)(93). In support of this proposal, an UVresonance Raman study found that significantconformational changes occurred at the Nterminus of sGC upon activator binding (96).

In sGC, the breaking of the proximal iron(Fe)-His bond is an essential event in theactivation by NO, and thus, a structure of afive-coordinate H-NOX protein would be veryimportant. Although a five-coordinate NOcomplex has not yet been determined, there aretwo recently solved structures where the neteffect is the severing of the proximal Fe-Hisbond; the solution structure of a S. oneidensis H-NOX mutant (94) and the crystal structure ofBAY 58-2667-bound Nostoc sp. H-NOX (101).Wild-type S. oneidensis H-NOX was shown tomodulate the activity of a histidine kinase ina ligand-dependent manner: Kinase activityis inhibited by the FeII-NO H-NOX but notthe FeII-unligated H-NOX (88). The H103GFeII-CO mutant mimics the kinase-inhibitoryactivity of the wild-type FeII-NO H-NOX (94).Because H103 is the proximal iron ligand in


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S. oneidensis H-NOX, mutation of this residueto glycine leads to the isolation of apoprotein.Similar to previous reports with other hemeproteins (92, 102), heme binding in S. oneidensisH-NOX H103G can be rescued by imidazole.H103 is in α-helix F, and in the heme-bindingrescued mutant, this helix is free to adopt aposition like that in a five-coordinate NOcomplex. The S. oneidensis H103G H-NOXstructure, along with the structure of theweakly active wild-type FeII-CO S. oneidensisH-NOX, was solved by NMR. The solutionstructures of both of these proteins indicatethat major changes in heme planarity andH-NOX conformation occur upon cleavage ofthe proximal histidine heme ligand.

Major structural changes are also observedbetween the unbound and BAY 58-2667-bound Nostoc sp. H-NOX structures (101).BAY 58-2667 is a NO- and heme-independentsGC activator (103) and is a candidate to treatdecompensated heart failure. The crystal struc-ture shows that BAY 58-2667 is able to displacethe heme and occupy the heme-binding site,thereby leading to a shift in the α-helix F thatcontains the proximal histidine residue. Addi-tional experiments mutating residues aroundthe proximal histidine on α-helix F suggest thatD102 could play a critical role in enzyme activa-tion (104). In addition to providing a structuralbasis for proposals on sGC activation, theseH-NOX structures enabled the developmentof homology models of the heme-bindingdomains of both NO-sensitive and atypical cy-clases (28, 63, 105, 106), as well as density func-tional theory analysis and computer simulationsto address questions concerning structuraldynamics (107, 108).

The crystal structure of a domain from theN. punctiforme signal transduction histidine ki-nase has also been determined (Figure 3). Thisdomain has high sequence identity (35%–38%)to the sGC PAS domain, and the crystal struc-ture showed that the domain dimerized andadopted a PAS fold (69). There is no struc-ture of a eukaryotic sGC PAS domain, but thecrystal structure of the coiled-coil domain ofthe β1 subunit has been solved (Figure 3) (31).

This structure indicates that the coiled-coil do-main forms a tetramer composed of a dimerof dimers. In addition to identifying potentialresidues involved in mediating dimerization,the authors propose that interhelix salt-bridgeformation selects for heterodimerization versushomodimerization in sGCs on the basis of theirstructure (31).

The catalytic domains of two different func-tional guanylate cyclases (78, 79), and the in-active human β1β1 catalytic domains [ProteinData Bank (pdb) code 2WZ1], have been struc-turally elucidated (Figure 3). The functionalcyclase domains include a soluble homodimericguanylate cyclase from the eukaryotic algaeC. reinhardtii (78) and a particulate homo-dimeric guanylate cyclase from the unicellularcyanobacterium Synechocystis PCC6803 Cya2(79). Both proteins cyclize GTP and likelycontain two catalytic sites per dimer. On thebasis of kinetic analysis of cGMP synthesis, theeukaryotic algae catalytic domains exhibit pos-itive cooperativity with a Hill coefficient of 1.5.This is similar to the cooperativity observedin particulate guanylate cyclases and may be acommon feature of homodimeric sGCs (78).The α1β1 heterodimer contains one active siteand a proposed pseudosymmetric site. Thisproposed pseudosymmetric site is thoughtto constitute an allosteric nucleotide-bindingsite that communicates with the catalyticnucleotide-binding site. On the basis of thealgae sGC structure, this communication mayinvolve residues contained on the β2-β3 loopof each catalytic domain monomer. Specifically,the interaction of D527 or E523 (C. reinhardtiisGC numbering) with a nucleotide in oneactive site could alter the loop conformationand thus lead to a conformational change inthe other nucleotide-binding site (78).

Unfortunately, both structures are in aninactive state, but they confirm the guanylatecyclase residues critical to metal binding andnucleotide recognition that were predictedfrom the adenylate cyclase crystal structures.Together, the reports of the C. reinhardtiisGC and Cya2 catalytic domains provided thefirst structures of a guanylate cyclase, and the


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structures show that there is high homologybetween guanylate cyclase and adenylatecyclase domains; however, the elucidation ofa mammalian heterodimeric structure remainsan important task for understanding sGCregulation. Details about how movement inthe β1 H-NOX domain affects the catalyticdomain, and the role of the PAS and coiled-coildomain in relaying a signal from the H-NOXdomain, may remain open questions until thefull-length sGC structure is elucidated.

LIGAND SELECTIVITY

The α1β1 heme environment is unique whencompared to the globins because the cofactorefficiently binds NO while having no affinityfor O2. Additionally, the α1β1 heterodimer ex-hibits an extremely slow rate of oxidation andhas among the highest midpoint potential re-ported for a high-spin heme protein (+187 mVversus +58 mV for myoglobin) (109). This abil-ity of mammalian sGC to select against O2

binding is important for it to function as a NOsensor, as O2 is present at much higher levelsthan NO in vivo, and FeII-O2 proteins reactrapidly with NO. Hemoproteins in the FeIII

oxidation state also form weak NO complexesand could inadvertently serve as a sink for NO.Since the discovery of sGC, several potentialmechanisms of ligand discrimination againstO2 have been proposed, including a weak Fe-His bond strength, a negatively charged distalpocket, and a sterically constrained distal pocket(109–111). One proposal on the mechanism bywhich α1β1 excludes O2 is based on analysis ofthe T. tengcongensis H-NOX crystal structure(63, 64). T. tengcongensis H-NOX stabilizes O2

binding with a hydrogen-bonding network in-volving a tyrosine (Y140), a tryptophan (W9),and an asparagine (N74) (Figure 4). Theseresidues appear to be absent in the β1 H-NOXprotein and other O2-excluding H-NOXs onthe basis of primary sequence alignments andhomology modeling. Conversely, several atyp-ical sGCs, including the known O2-binding cy-clase Gyc-88E, encode a tyrosine that alignswith T. tengcongensis H-NOX Y140. Therefore,

the absence of a hydrogen bond donor in theα1β1 distal heme pocket, and the subsequentincrease in the O2 off rate, likely contributesto the protein’s inability to form a stable FeII-O2 complex (64, 90). Molecular dynamics simu-lations of O2-binding and non-O2-binding H-NOX proteins suggest that they have varyingtunnel systems, which may also contribute totheir different ligand-binding properties (112),and site-directed mutagenesis within the pro-posed tunnel indicates that it is important fordiffusion of gaseous ligands (113).

Several biochemical studies aimed atprobing ligand selectivity in sGC have beenreported. The distal-pocket tyrosine in theO2-binding T. tengcongensis H-NOX wasmutated to leucine (Y140L), and this mutationsignificantly reduced O2 affinity. Additionally,the introduction of a distal-pocket tyrosine in anon-O2-binding H-NOX from Legionella pneu-mophilia (L2 H-NOX) enabled the protein tobind O2 (90). Mutagenesis studies introducinga tyrosine into the distal pocket of the β1 H-NOX-PAS domain β1(1–385) also produced aprotein that was capable of binding O2 (90), butthe same mutation in full-length sGC did notfacilitate O2 binding (106, 114). However, theintroduction of a tyrosine and glutamine intothe β1 heme pocket (I145Y/I149Q) resulted ina full-length α1β1 protein with altered reactiv-ity to O2 (105). A homology model of the O2-binding guanylate cyclase Gyc-88E places theseamino acids within the predicted distal hemepocket. Thus, some O2-binding guanylate cy-clases may utilize a Tyr/Gln hydrogen-bondingnetwork, similar to several truncated globins(115–118), to stabilize ligand binding. Themarked variability in O2 reactivity between full-length sGC and heme-binding truncations ofthe β1 protein highlights the potential for othersGC domains to influence ligand selectivity.Thus, despite significant progress in our under-standing of ligand discrimination in sGC, in-cluding the identification of residues critical forstabilizing O2 binding in sGC homologs, thereremain some unknown variables that may con-tribute to the ligand specificity of these hemeproteins.


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REGULATION OF SOLUBLEGUANYLATE CYCLASE BYNITRIC OXIDE

Physiological responses to NO, such as smoothmuscle relaxation, are rapidly induced by lowlevels of the diatomic gas. In cells, cGMPlevels rise within milliseconds after exposureto nanomolar concentrations of NO, and thisfast response occurs because sGC efficientlybinds to and is activated by NO. When NOdissociates from the protein, the amount ofcGMP decreases to a basal level. Thus, boththe rise and fall of cGMP levels must be tightlyregulated for proper function of cGMP-dependent processes. Upon repeated exposureto NO or NO-donors, like GTN, maximalsGC activation decreases, and this process iscalled sGC desensitization. sGC activation,deactivation, and desensitization have beenextensively studied in vivo and in vitro. A majorchallenge with understanding these processesoccurs when reconciling discrepancies inresults obtained with purified protein versussGC examined in the cellular milieu.

Activation and NitricOxide Association

As mentioned above, the activation of sGCin vitro and in vivo is rapid (occurs in mil-liseconds). Using a NO donor or NO gas,the apparent 50% effective concentration forNO acting on purified sGC is between 80 and250 nM (119, 120). A similar potency of NOhas been measured in rat cerebellar cells (ap-parent 50% effective concentration ∼45 nM)(121). On the basis of studies with purifiedprotein, it is known that NO binds to the hemeof sGC at a diffusion-controlled rate to form aninitial six-coordinate complex, which rapidlyconverts to a five-coordinate ferrous nitrosyl(FeII-NO) complex (120). The six-coordinateFeII-NO complex is not stable and has onlybeen observed with time-resolved spectro-scopic methods (120, 122, 123). The presenceof Mg2+GTP or Mg2+/cGMP/pyrophosphate(PPi) accelerates the formation of the

five-coordinate sGC-NO complex, andthis effect is blocked by the addition of ATP(122, 123).

Breakage of the Fe-His bond is thought tobe a critical step in the activation of sGC byNO; however, recent data have shown that NOcoordination to the heme is not sufficient forfull activation ex vivo (122, 123). A low-activityFeII-NO complex can be formed in the pres-ence of stoichiometric amounts of NO, whereasa high-activity FeII-NO complex is formed inthe presence of excess NO. These two five-coordinate FeII-NO species are indistinguish-able by electronic absorption spectroscopy, butthey exhibit distinct signals by electron para-magnetic spectroscopy (124). Preincubation ofsGC with substrate Mg2+GTP or the reactionproducts Mg2+/cGMP/PPi produces a high-activity FeII-NO species in the presence ofstoichiometric amounts of NO (123). ATP cancompete with the GTP effect, and in the pres-ence of ATP and GTP, a low-activity FeII-NOspecies is formed (122). This clear differencein sGC activity indicates that preincubationof the enzyme with the small molecules leadsto a conformational change such that sGC ishighly activated by low levels of NO. It wasalso found that the small-molecule YC-1 canactivate the low-activity FeII-NO complex tothe high-activity state (122, 125). Thus, bothYC-1 and excess NO activate the low-activityfive-coordinate FeII-NO complex that isformed in the absence of substrate or reactionproducts.

Two mechanisms have been proposed to ac-count for the varying activity of the sGC FeII-NO complex observed ex vivo. One proposalis that excess NO activates the FeII-NO com-plex by binding to nonheme sites on the pro-tein (122). If NO binds to a nonheme site,it is likely that cysteines would comprise thisbinding site as experiments with the thiol reac-tive reagent methyl methanethiosulfonate sug-gest that reduced cysteines are necessary for themechanism of NO activation (125). The secondproposal regarding sGC activation involves ex-cess NO binding to the heme to form a tran-sient dinitrosyl complex, which then converts


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Preincubation with GTP increases rate

ATP inhibits this GTP effect

In vitro measurements

NO dissociation

Deactivation

YC-1 decreases rate

No effect with GTP or ATP

FeII

His

NO

FeII

His

NO

FeII

His

FeII

His

NO

YC-1 or BAY 41-2272 increases rate399 nm 399 nm 431 nm

High activity

FeII

His

NO

Low activity

Slow Slow

Fast

NO

Figure 5Model of nitric oxide (NO) dissociation and deactivation of NO-stimulated soluble guanylate cyclase (sGC)in vitro. The sGC-NO complex consists of two different five-coordinate species that slowly interconvert.NO dissociation from the sGC heme is slow, whereas NO deactivation is rapid on the basis of in vitromeasurements, including spectroscopic methods (Absmax indicated in nanometers) and cyclic GMP analysis.The presence of allosteric modulators (GTP, ATP, YC-1, or BAY 41-2272) influences the deactivation andNO dissociation rates differently. Taken together, these results indicate that the deactivated sGC species isdistinct from the unligated sGC species (431 nm) produced from NO dissociation from the heme andsuggests that two molecules of NO are able to bind to sGC.

to a five-coordinate complex with NO bound inthe proximal heme pocket (123). Such proximalheme pocket NO binding has been observedwith other histidine-ligated heme proteins in-cluding cytochrome c′ (126).

The importance of the two different sGC-NO states in vivo remains unclear. One reportexamining sGC activity in endothelial cellsproposed that enzyme activation by excess NOis important for the physiological activation ofsGC (125), whereas another report examiningsGC activation in rat platelets and cerebellarcells proposed that the observed activationkinetics were consistent with a single NO-binding event (127). Thus, further experimentsare necessary to resolve the mechanism of sGCactivation in vivo.

Deactivation and NitricOxide Dissociation

Deactivation of the sGC FeII-NO complexhas been extensively studied to understand the

lifetime of the NO signal in vivo. This processwas originally thought to correlate directly withNO dissociation from the sGC heme; however,as mentioned above, a NO-bound protein thatis partially activated has been characterized.This indicates that sGC deactivation and NOdissociation from the heme cofactor are notexplicitly linked, and thus, deactivation andheme-NO dissociation measurements cannotbe used interchangeably (Figure 5).

NO-induced relaxation of smooth musclecells dissipates within seconds upon removal offree NO due to the deactivation of sGC (128).sGC deactivation, the rate of decline in cGMPsynthesis after an activator is removed, hasbeen determined in various cells and with puri-fied protein. In cells, sGC deactivation is rapid(t1/2 < 5 s at 37◦C) in the presence of an NO trap(oxyhemoglobin) to limit NO rebinding (121,129). Activity assays with the cytosol of retinahomogenate indicate that the sGC-NO deac-tivation rate is not influenced by the presence


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S-nitrosation:posttranslationalmodification involvingthe oxidative additionof NO to a thiol

of Mg2+GTP and is only slightly increased bythe presence of reducing agents, such as glu-tathione and dithiothreitol (129). In agreementwith cellular data, the deactivation rate of pu-rified protein is also rapid (t1/2 ∼ 4 s at 37◦C)(130). This deactivation rate is unaffected by thepresence of a GTP analog and/or ATP (122),but the rate is significantly decreased (140-fold)by the presence of the allosteric activator YC-1(t1/2 > 10 min at 37◦C) (130).

The rate of NO dissociation from the sGCheme must be spectroscopically determined,and as a consequence, this rate has only beenmeasured in vitro. Early reports found thatthe sGC heme-NO complex is very stable(t1/2 ∼ 87 min at 37◦C) in the absence of NOtraps or allosteric effectors (23, 131). Unlikemost heme proteins, which rapidly oxidize inthe presence of O2 and NO, a reduced FeII

protein is formed when NO dissociates fromthe sGC heme. Using a trap to scavenge NO,like CO/dithionite or oxymyoglobin, the dis-sociation rate can be determined without in-terference from NO rebinding. Reductants likedithiothreitol or glutathione react with NO andcan also be used to prevent rebinding (23). Withthese NO trapping methods, it was determinedthat the NO dissociation rate is relatively slow(t1/2 = 3–8 min at 37◦C) (23, 27, 131) com-pared to previously determined deactivationrates but increases (∼50-fold) in the presenceof Mg2+GTP (131). However, if Mg2+GTP isadded to sGC after the NO complex is formed,it has no effect on the dissociation rate (23, 123,131), indicating that the allosteric affect of GTPis dependent on substrate binding in the ab-sence of NO. In contrast to the previously dis-cussed deactivation results, the GTP effect onthe dissociation rate is inhibited by the pres-ence of ATP (122). Therefore, in the presenceof both GTP and ATP, sGC exhibits a slowNO dissociation rate but rapid deactivation. Inthe presence of YC-1, the NO dissociation rateincreases (27), whereas the deactivation rate de-creases (130). A model summarizing deactiva-tion of sGC and NO dissociation is shown inFigure 5.

Desensitization

The sGC response to GTN or NO decreasesupon repeated exposure to the activators. Thisdesensitization is rapid, such that a singlepretreatment of cells with GTN or NO cansignificantly reduce cGMP stimulation uponthe second exposure (132, 133). Purifiedprotein in the absence of reducing agents alsoexhibits a decrease in NO-stimulated activityafter pretreatment with NO. This desensiti-zation has implications for therapies used inthe treatment of heart disease as this loss ofresponsiveness, or tolerance, is also observedwhen organic nitrates are administered topatients with angina pectoris (134). As a conse-quence, these drugs cannot be used repeatedly.Over the past 30 years, several proposals havebeen advanced to explain the phenomenon oftolerance, including increased PDE activity(135), inhibition of mitochondrial aldehydedehydrogenase (136), and most recentlyS-nitrosation (137, 138). There is currentlysignificant evidence in support of the proposalthat nitrosation, the oxidative addition of NOto a thiol, contributes to sGC desensitization.

It has long been known that thiol oxidationinhibits sGC activity. Cysteines can oxidizesequentially to form a sulfenic acid, sulfinicacid, sulfonic acid, or a disulfide bond in thepresence of O2, or form a nitrosothiol in thepresence of NO and O2. In the 1980s, the for-mation of sGC-cysteine mixed disulfides wasshown to reversibly inhibit cGMP production(139). More recently, the induction of reactiveO2 species in vascular smooth muscle cells wasfound to inhibit cGMP synthesis and lead to theoxidative modification of sGC cysteines (140).Additionally, the thiol modifying reagentmethyl methanethiosulfonate was shown toinhibit sGC activity in vitro and in primaryendothelial cells. In vitro sGC inhibition bymethyl methanethiosulfonate is reversible, andthe molecule was shown to modify severalcysteines on both the α1 and β1 subunits(125). The NO-dependent oxidation of cys-teines on sGC has also been reported. sGCis S-nitrosated in the presence of low levels


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Nitrate tolerance:loss of sensitivity tonitrates

of NO, and this modification has been linkedto a reduction in NO-stimulated activity(137). In addition, GTN has been shown toinduce sGC S-nitrosation and desensitizationin a concentration-dependent manner (138).Cysteines on both the α1 (C243) and β1(C122) subunits have been identified as targetsof this oxidative modification.

It has become clear that sGC requires freethiols for proper function. Without a crystalstructure, it is difficult to address why these cys-teines are necessary for NO-induced sGC acti-vation. Perhaps free thiols are important to thestructural integrity of the protein, involved in aconformational change to the activated enzymestate, and/or directly involved in the NO mech-anism of activation. If sGC S-nitrosation is theprimary molecular mechanism of nitrate toler-ance, then molecules developed to protect thesethiols from oxidation could be useful for thetreatment of diseases related to sGC dysfunc-tion. Furthermore, the apo- and heme-oxidizedsGC states have been proposed to be physiolog-ically relevant sGC species in diseased tissue.

MODULATORS OF SOLUBLEGUANYLATE CYCLASE ACTIVITY

Soluble Guanylate Cyclase Activators

sGC is a therapeutic target in the treatmentof heart disease. Organic nitrites and organicnitrates (like GTN) are perhaps the earliestagents used to target the NO/cGMP-signalingpathway and have been in clinical use for over100 years. The first description of GTN as atherapeutic agent for the treatment of anginapectoris appeared in 1879, and it remains thedrug of choice to treat the disease. Despitedecades of clinical use, the precise mechanismof action of GTN is unknown; however, itis generally considered a nitrovasodilator ora NO-donor. This NO release may occur byspontaneous decomposition or bioconversionto result in NO-dependent sGC activation.

In addition to NO, other known heme lig-ands, including CO, nitrosoalkanes, and alkylisocyanides, can bind to the sGC heme but can

only weakly activate the protein (17, 141, 142).The binding of these compounds leads to theformation of a six-coordinate complex and to atwo- to fourfold increase in the rate of cGMPproduction, significantly lower than the 100- to400-fold increase in cGMP synthesis observedwith NO. Other compounds that have been re-ported to activate sGC by targeting the heme-binding pocket include protoporphyrin IX (15)and Co2+ protoporphyrin IX (143).

It has become clear that small moleculescan modulate the activity of sGC and thatnew therapeutics might be developed for thetreatment of various diseases. This prompteda search for novel sGC activators, and severalcompounds were screened for the ability to in-crease cGMP levels in cell lysates. Such a screenled to the identification of YC-1, a benzylin-dazole derivative that activates sGC withoutcoordinating to the heme (144). YC-1 onlyactivates the FeII-unligated sGC state two- tofourfold but significantly increases sGC activitywhen a ligand is bound at the FeII heme (141,142, 145, 146). This synergistic activation leadsto an FeII-CO complex that is activated 100-to 400-fold and an FeII-NO complex that isactivated 200- to 800-fold. The molecularmechanism of YC-1 activation is unknown.Experiments with equilibrium dialysis suggestthat sGC binds one equivalent of YC-1 perheterodimer (147). This binding site maybe contained within the N terminus of theα1 subunit (33, 148) or within the pseu-dosymmetric substrate site (149–151). Whatis clear is that YC-1 binding to sGC inducesa conformational change that elevates cGMPproduction. This highly active conformationalstate has been characterized by spectroscopicmethods, including electronic absorption(152), resonance Raman spectroscopy (147,153), and electron paramagnetic resonancespectroscopy (124, 147). There are also clearkinetic effects of adding YC-1 to CO- orNO-bound sGC (27, 130, 154).

The discovery of the novel sGC stimulatorYC-1 led several groups to carry out structureactivity relationships to improve both the sol-ubility and efficacy of YC-1. With this work


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came the identification of several other com-pounds, including BAY 41-2272, BAY 41-8543,CMF-1571, and A-350619 (reviewed in Refer-ence 103). Although there is some debate overthe possible inhibition of PDE5 by BAY 41-2272 (155, 156), it is commonly accepted thatthe molecule activates sGC without coordinat-ing to the heme. Collectively, these moleculesconstitute a novel class of sGC stimulatorsthat require the presence of the heme moietyand have the ability to synergistically stimulatesGC with both NO and CO. These moleculeswere promising drug candidates but had un-favorable drug metabolism. Medicinal chem-istry efforts to reduce the problems associatedwith these compounds led to the discovery ofBAY 63-2521 (riociguat). This compound in-duces vasodilation by activating sGC and is cur-rently in Phase III clinical trails (reviewed inReference 157).

There are also small-molecule activatorsthat target heme-oxidized or -deficient sGC;therefore, they are classified as NO and hemeindependent. One of these is BAY 58-2667(Cinaciguat) (158). BAY 58-2667 is generallycited as an activator of both heme-deficientand heme-oxidized (FeIII) sGC (158), but thereis one report that proposes the molecule ex-clusively targets the heme-deficient sGC state(159). BAY 58-2667 is proposed to activate sGCby binding within the heme pocket, therebyserving as a mimic of the heme-NO complex(91, 101). In addition to activating the en-zyme, BAY 58-2667 also stabilizes sGC andprotects the protein from degradation (160). Inrats, BAY 58-2667 was shown to lower bloodpressure (161) and protect against ischemic in-jury (162), and the compound has been testedon patients with acute decompensated heartfailure (163–165).

Soluble Guanylate Cyclase InhibitorsThere has been significantly more work onidentifying sGC activators, but compoundsthat inhibit sGC activity have also been re-ported. These compounds are not as selec-tive as the identified activators and includegeneral oxidants, molecules that target hemo-proteins, and nucleotide cyclase inhibitors.Compounds proposed to target the sGC H-NOX domain include heme, hematin (15),and 1H-[1,2,4]oxadiazolol[4,3-a]quinoxalin-1-one (ODQ) (166). These compounds inhibitsGC by oxidation of the ferrous iron inthe heme cofactor. Molecules such as hy-drogen peroxide, superoxide, cystine, and S-nitrosocysteine also reduce cGMP productionby oxidizing critical residues on sGC (138–140). Other known sGC inhibitors, such asLY83583 (167) and methylene blue (168), in-hibit the enzyme via generation of super-oxide anion (169, 170). Inhibition of sGCby these oxidants emphasizes the importanceof the redox environment for proper proteinfunction.

Substrate analogs that bind to the sGCcatalytic domain inhibit guanylate cyclaseactivity. To date, several such analogs havebeen identified as sGC inhibitors, including,but not limited to, ITP, XTP, CTP, ATP,ADP, AMP, 2′-deoxyadenosine 5′-diphosphate(2′-dADP), GDP, guanosine-5′-[(α,β)-methylene]triphosphate (GMP-CPP), andN-methylanthraniloyl (MANT)-nucleotides(75, 151, 171, 172). These compounds inhibitsGC by varying mechanisms depending onwhether they target the pseudosymmetric sitein addition to the substrate-binding site. Forexample, AMP-PNP is a competitive inhibitor(151), while ATP is a mixed-type inhibitor andsubstrate (76).

SUMMARY POINTS

1. Soluble guanylate cyclase (sGC) is the most thoroughly characterized receptor for thesignaling molecule nitric oxide (NO). NO activation of sGC is essential to several physi-ological processes, and thus dysfunction in sGC is linked to several diseases. Compoundsthat modulate sGC are in clinical trails for the treatment of heart disease.


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2. Each sGC subunit consists of an H-NOX, PAS, coiled-coil, and catalytic domain. Func-tional truncations of sGC include the isolated β1 H-NOX domain, the β1 coiled-coildomain, and the α1β1 catalytic domains.

3. Genome mining has revealed that several predicted proteins exist with high sequencehomology to sGC, including prokaryotic H-NOXs and eukaryotic NO-sensitive andatypical sGCs.

4. The structure of full-length sGC is not yet determined, but studies on H-NOXs and sGChomologs have illuminated many important structural features of the enzyme. On thebasis of these studies, sGC likely contains a hydrophobic heme-binding pocket, withouta distal-pocket hydrogen bond donor, and heterodimerization is mediated by contactson the PAS, coiled-coil, and catalytic domains.

5. α1β1 sGC does not form a stable complex with O2, but several sGC homologs bind O2.Hydrogen bond donors in the heme-distal pocket are often found in the H-NOXs andatypical sGCs that bind O2. The presence or absence of amino acids capable of forminga hydrogen bond in the heme-distal pocket contributes to selectivity for gaseous ligands.

6. sGC regulation by NO and nucleotides in vitro is complex. Two molecules of NO canbind to the protein, and both GTP and ATP can influence NO binding, dissociation,and activation. In cells, it is likely that NO, GTP, and ATP modulate cGMP production,and both reduced cysteines and a reduced heme iron are important for this activity.

FUTURE ISSUES

Over the past few years, sGC has been implicated in an expanding number of physiologi-cal processes and diseases. Continued progress toward elucidating the mechanism of sGCactivation is important in the development of therapeutics to treat disorders relating tothe NO-signaling pathway. Despite significant advances in our understanding of NO asa signaling agent, many questions remain unanswered. In mammals, the sGC response toNO is complicated, and the precise events that lead to activation remain a topic of debate.Furthermore, sGC is regulated by allosteric interactions with ATP and GTP, and there arelikely other important factors, including S-nitrosation and phosphorylation, that modulatesGC activity. Future experiments considering these factors will be necessary to determinethe influence of each regulatory factor in the activation, deactivation, and desensitization ofsGC. How do the α1 and β1 subunits interact when sGC is activated? What is the natureof the allosteric activator-binding site? What conformational changes occur when ligandsbind to sGC? All of these questions may be addressed with the structural elucidation of thefull-length heterodimeric protein. Additionally, structural analysis may provide insight intothe molecular mechanisms that lead to sGC dysfunction.

DISCLOSURE STATEMENT

M.A.M. is a cofounder of Omniox, Inc., a company commercializing oxygen delivery technologyfor a broad range of clinical indications in cancer, surgical, and cardiovascular markets. The authorsare not aware of any other affiliations, memberships, funding, or financial holdings that might beperceived as affecting the objectivity of this review.


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ACKNOWLEDGMENTS

This article was written while M.A.M. was employed by the University of California, Berkeley.The authors acknowledge members of the Marletta lab, past and present, who have contributed tostudies on sGC, NOS, and H-NOXs. Research on sGC in the authors’ laboratory was supportedby the National Institutes of Health (NIH grant GM077365), and research on H-NOXs by NIHgrant GM070671.

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BI81-FrontMatter ARI 9 May 2012 7:26

Annual Review ofBiochemistry

Volume 81, 2012Contents

PrefacePreface and Dedication to Christian R.H. Raetz

JoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �xi

Prefatories

A Mitochondrial OdysseyWalter Neupert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Fires of LifeGottfried Schatz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �34

Chromatin, Epigenetics, and Transcription Theme

Introduction to Theme “Chromatin, Epigenetics, and Transcription”Joan W. Conaway � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �61

The COMPASS Family of Histone H3K4 Methylases:Mechanisms of Regulation in Developmentand Disease PathogenesisAli Shilatifard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

Programming of DNA Methylation PatternsHoward Cedar and Yehudit Bergman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �97

RNA Polymerase II Elongation ControlQiang Zhou, Tiandao Li, and David H. Price � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 119

Genome Regulation by Long Noncoding RNAsJohn L. Rinn and Howard Y. Chang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 145

Protein Tagging Theme

The Ubiquitin System, an Immense RealmAlexander Varshavsky � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 167

Ubiquitin and Proteasomes in TranscriptionFuqiang Geng, Sabine Wenzel, and William P. Tansey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 177

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The Ubiquitin CodeDavid Komander and Michael Rape � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 203

Ubiquitin and Membrane Protein Turnover: From Cradle to GraveJason A. MacGurn, Pi-Chiang Hsu, and Scott D. Emr � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 231

The N-End Rule PathwayTakafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, and Yong Tae Kwon � � � 261

Ubiquitin-Binding Proteins: Decoders of Ubiquitin-MediatedCellular FunctionsKoraljka Husnjak and Ivan Dikic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

Ubiquitin-Like ProteinsAnnemarthe G. van der Veen and Hidde L. Ploegh � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 323

Recent Advances in Biochemistry

Toward the Single-Hour High-Quality GenomePatrik L. Stahl and Joakim Lundeberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 359

Mass Spectrometry–Based Proteomics and Network BiologyAriel Bensimon, Albert J.R. Heck, and Ruedi Aebersold � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 379

Membrane Fission: The Biogenesis of Transport CarriersFelix Campelo and Vivek Malhotra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Emerging Paradigms for Complex Iron-Sulfur Cofactor Assemblyand InsertionJohn W. Peters and Joan B. Broderick � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 429

Structural Perspective of Peptidoglycan Biosynthesis and AssemblyAndrew L. Lovering, Susan S. Safadi, and Natalie C.J. Strynadka � � � � � � � � � � � � � � � � � � � � 451

Discovery, Biosynthesis, and Engineering of LantipeptidesPatrick J. Knerr and Wilfred A. van der Donk � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 479

Regulation of Glucose Transporter Translocationin Health and DiabetesJonathan S. Bogan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 507

Structure and Regulation of Soluble Guanylate CyclaseEmily R. Derbyshire and Michael A. Marletta � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

The MPS1 Family of Protein KinasesXuedong Liu and Mark Winey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 561

The Structural Basis for Control of Eukaryotic Protein KinasesJane A. Endicott, Martin E.M. Noble, and Louise N. Johnson � � � � � � � � � � � � � � � � � � � � � � � � � � 587

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Measurements and Implications of the Membrane Dipole PotentialLiguo Wang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 615

GTPase Networks in Membrane TrafficEmi Mizuno-Yamasaki, Felix Rivera-Molina, and Peter Novick � � � � � � � � � � � � � � � � � � � � � � � 637

Roles for Actin Assembly in EndocytosisOlivia L. Mooren, Brian J. Galletta, and John A. Cooper � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 661

Lipid Droplets and Cellular Lipid MetabolismTobias C. Walther and Robert V. Farese Jr. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 687

Adipogenesis: From Stem Cell to AdipocyteQi Qun Tang and M. Daniel Lane � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 715

Pluripotency and Nuclear ReprogrammingMarion Dejosez and Thomas P. Zwaka � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 737

Endoplasmic Reticulum Stress and Type 2 DiabetesSung Hoon Back and Randal J. Kaufman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 767

Structure Unifies the Viral UniverseNicola G.A. Abrescia, Dennis H. Bamford, Jonathan M. Grimes,

and David I. Stuart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 795

Indexes

Cumulative Index of Contributing Authors, Volumes 77–81 � � � � � � � � � � � � � � � � � � � � � � � � � � � 823

Cumulative Index of Chapter Titles, Volumes 77–81 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 827

Errata

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

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AnnuAl Reviewsit’s about time. Your time. it’s time well spent.

AnnuAl Reviews | Connect with Our expertsTel: 800.523.8635 (us/can) | Tel: 650.493.4400 | Fax: 650.424.0910 | Email: [email protected]

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Annual Review of Statistics and Its ApplicationVolume 1 • Online January 2014 • http://statistics.annualreviews.org

Editor: Stephen E. Fienberg, Carnegie Mellon UniversityAssociate Editors: Nancy Reid, University of Toronto

Stephen M. Stigler, University of ChicagoThe Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the field of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specific application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences.

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•Brain Imaging Analysis, F. DuBois Bowman•Statistics and Climate, Peter Guttorp•Climate Simulators and Climate Projections,

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Variable Models, David M. Blei•Structured Regularizers for High-Dimensional Problems:

Statistical and Computational Issues, Martin J. Wainwright

•High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier

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Statistical Issues, Harvey Goldstein•Statistical Ecology, Ruth King•Estimating the Number of Species in Microbial Diversity

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Hong Qian, S.C. Kou•Statistics and Quantitative Risk Management for Banking

and Insurance, Paul Embrechts, Marius Hofert

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