NOSICVW
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`AMERICAN
`ASSOCIATION FOR THE
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`SCIENCE ISSN 0036-8075
`
`18 JANUARY 1991
`VOLUME 251
`NUMBER 4991
`
`Editorial
`
`247
`This Week in Science
`
`
`249
`Teaching and Research
`
`256 Safety of Bovine Growth Hormone:D.S. KRONFELD; J. C. JUSKEVICH AND
`C. G. Guyer @ Interpreting Cancer Tests: J. D. WILson; G. W. GRIBBLE &
`Kidney Transplantation: Overlooked Pioneer: G. B. ELION
`LNews&Commentim
`The Rush to Publish @ Lessons from Physics
`Third Strike for Idaho Reactor
`CDC AbandonsPlans for AIDS Survey
`Healy Nominated
`265
`GAO and DODGetInto a Cat Fight
`266
`Science Literacy: The Enemy Is Us @ Science’s Top 20 Greatest Hits
`268
`New Light on Writing in the Americas
`271
`Montagnier Pursues the Mycoplasma-AIDS Link
`272
`Despite Reports of Its Death, the Big Bang Is Safe
`274
`Global Temperature Hits Record Again
`275
`Briefings: Radiation Research Shake-Up m Private Initiative on Fetal Research @
`U.K. Antes Upfor Telescopes @ George Mason to Set Up Think Tank
`
`277
`Subsistence EconomyofEl Paraiso, an Early PeruvianSite: J. QUILTER,
`B. Oyepa E., D. M. PEARSALL, D. H. SANDWEISS, J. G. JONES, E. S$. WING
`Chemistry and Biology of the Immunophilins and Their Immunosuppressive
`Ligands: $. L. SCHREIBER
`
`
`_ResearchArticle [ir
`CCAAT-Enhancer Binding Protein: A Componentof a Differentiation Switch:
`R. M. Umekx,A. D. FrrepMan,S. L. MCKNiGHt
`
`293 An Antimony Sulfide with a Two-Dimensional, Intersecting System of Channels:
`J. B. Parise
`
`263
`264
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`283
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`COVER
`Lakes and ponds on the arctic tundra with Itigaknit Mountain in the
`background, North Slope, Alaska. These aquatic ecosystems are continuously
`releasing carbon dioxide to the atmosphere. Much of the carbon originates in
`terrestrial environments, and accounting for this release substantially lowers the
`estimate ofthe worldwide arctic sink for atmospheric carbon dioxide. See page 298.
`[Photograph by George W.Kling]
`
`
`
`301
`
`294 Local Structure and Chemical Shifts for Six-Coordinated Silicon in High-Pressure
`Mantle Phases: J. F. STEBBINS AND M. KANZAKI
`298 Arctic Lakes and Streams as Gas Conduits to the Atmosphere: Implications for
`Tundra Carbon Budgets: G. W. Kiina, G. W. Kippnut, M. C. MILLER
`Putative Skeletal Neural Crest Cells in Early Late Ordovician Vertebrates from
`Colorado: M. M. SMITH
`303 Altered Perception of Species-Specific Song by Female Birds After Lesions of a
`Forebrain Nucleus: E. A. BRENOWITZ
`305 The Effect of Anti-Neoplastic Drugs on Murine Acquired Immunodeficiency
`Syndrome: C. SIMARD AND P, JOLICOEUR
`308 Evidence for Biased Gene Conversion in Concerted Evolution of Ribosomal DNA:
`D. M. Hixiis, C. Morrrz, C. A. Porter, R. J. BAKER
`310 The Effect of the Floor Plate on Pattern and Polarity in the Developing Central
`Nervous System: $. H1kANO, S. Fuse, G. S. SOHAL
`313 Regulation of Interleukin-2 Gene Enhancer Activity by the T Cell Accessory
`Molecule CD28: J. D. Fraser, B. A. InvinG, G. R. CRABTREE, A. WEISS
`
` Microwave Sounding Units and Global Warming: B. L. Gary AND S.J. Ker;
`
`Protein Immunoblotting Incubation Rotator @ Micromanipulator Table @ Leiden
`Microincubator @ Freezing Stage with Microtome @ Data Analysis for the
`Macintosh ® Monoclonal Antibodies ® Literature
`
`
`
`Technical Comments
`
`Book Reviews
`
`R. W. SreENCER ANDJ, R. Curisty ® Lipid Flow in Locomoting Cells:
`M.S. BrerscHER; K. JAcopson, J. LEE, M. Gusrarsson, K.-E. MAGNUSSON
`Bryozoan Morphological and Genetic Correspondence: What Does It Prove?:
`J. Levinton; J. B. C. JACKSON AND A. H. CHEETHAM
`
`Authors of Their Own Lives, reviewed by A. Sica @ Australian Ecosystems,
`M. LowMaN ® Thalamic Oscillations and Signaling AND Brainstem Control of
`Wakefulness and Sleep, C. Koco ™ Books Received
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`Editorial Board
`Board of Directors
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`Treasurer
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`18 JANUARY 1991
`
`Board of Reviewing
`Editors
`John Abelson
`Frederick W. Alt
`Don L. Anderson
`Stephen J. Benkovic
`Gunter K-J Blobel
`Floyd E. Bloom
`Henry R. Bourne
`James J, Bull
`Kathryn Calame
`Charles R. Cantor
`Ralph J. Cicerone
`John M. Coffin
`Robert Dorfman
`Bruce F. Eldridge
`Paul T. Englund
`Fredric S, Fay
`
`
`Dennis A. Powers
`Erkki Ruoslahti
`Thomas W. Schoener
`Ronald H. Schwartz
`Terrence J. Sejnowski
`Thomas A. Steitz
`Robert T. N. Tijan
`Emil R. Unanue
`Geerat J. Vermeij
`Bert Vogelstein
`Harold Weintraub
`Zena Werb
`George M. Whitesides
`Owen N. Witte
`William B. Wood
`Keith Yamamoto
`
`Harry A. Fozzard
`Theodore H, Geballe
`Roger |. M, Glass
`Stephen P. Goff
`Corey 5. Goodman
`Stephen J. Gould
`Eric F. Johnson
`Stephen M. Kosslyn
`Konrad B. Krauskopf
`Charles S. LevingsIll
`Richard Losick
`John G. McGiff
`Anthony R, Means
`Mortimer Mishkin
`RogerA. Nicoll
`William H. Orme-Johnson Ill
`Carl O, Pabo
`Yeshayau Pocker
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`TABLE OF CONTENTS
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`245
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`BE. P. Lanning, Peru Before the Incas (Prentice-Hall, Englewood Cliffs, NJ, 1967),
`4.
`5. T, C. Patterson, in Prehistoric Agriculture, S. Streuver, Ed. (Natural History Press,
`Garden City, NY, 1971), pp. 181-208.
`6. R. Fung P., Apuntes Arqueol. 2, 11 (1972).
`7. J. H. Steward and L. C. Faron, Native Peoples ofSouth America (McGraw Hill, New
`York, 1959).
`8. M. H, Parsons, Am, Antig. 35, 292 (1970).
`9,
`1D. J. Wilson, Am. Anthropol. 83, 93 (1981).
`10.
`J, S. Raymond, Am. Antig. 46, 806 (1981).
`LL.
`J. Quilter and T. Stocker, Am. Anthropol, 85, 545 (1983).
`12.
`J. Quilter, J. Field Archaeol. 12, 279 (1985).
`13.
`FP. A. Engel, J. Soe. Am. 55, 43 (1966); Anal. Cient. Univ, Agraria 5, 241
`(1967).
`14. A. Osborn,in For Theory Building in Archaeology, L. Binford, Ed, (Academic Press,
`New York, 1977), pp. 157-243.
`15. S. Quilter suggested this possibility during work at the Paloma Site in 1976.
`16. C. E. Smith,
`in La Galgada Peru, A Preceramic Culture in Transition, Terence
`Grieder et al., Eds. (University of Texas Press, Austin, TX, 1988), pp. 125-151.
`17. 8. Pozorski and T. Pozorski, Early Settlement and Subsistence in the Casma Valley,
`Pern (University of Iowa Press, Iowa City, 1987).
`18. V. Popper, in Los Gavilanes, Mar, Desierto y Oudisis en la Historia del Hombre, D.
`Bonavia, Ed. (Editorial Ausonia, Lima, Peru, 1982), pp. 148-156,
`19. 5. Pozorski and T. Pozorski, Ann. Carnegie Mus. Nat. Hist. 49, 337 (1979); J. B.
`Bird, Anthropol. Pap. Am. Mus. Nat. Hist. 62 (1985), part 1.
`20. A. Grobman, in Los Gavilanes, Mar, Desierto y Odsis en la Historia del Hombre, D.
`
`24.
`
`Bonavia, Ed, (Editorial Ausonia, Lima, Peru, 1982), pp. 157-179; R. L. Burger
`and N. van der Merwe, Am. Anthropol. 92, 96 (1990).
`21. T. Dillehay, P. Netherly, J. Rossen, Am. Antig. 54, 733 (1989).
`22.
`J. Quilter, “Tofish in the afternoon: Beyond subsistence economies in the study of
`early Andeancivilization,” paper presented at 51st Annual Meeting of the Society
`for American Archacology, New Orleans, LA, 23 April 1986.
`23. M. Moseley, Pre-agricultural Coastal Civilizations in Peru (Carolina Biology Read-
`ers, no. 90, Burlington, NC, 1978).
`J. Quilter, “Core and periphery in Preceramic coastal Peru,” paper presented at the
`88th Annual Meeting of the American Anthropological Society, Washington, DC,
`19 November 1989,
`25. R. G, Wilkinson, Poverty and Progress (Praeger, New York, 1973).
`26. Following taxa listed in V. Alamo V. and V. Valdiviezo M., Bolet, Inst. Mar Peni
`(volumenextraordinario. Callao, Peru, 1987).
`27. Funding for the El Paraiso research was provided by NSF grant BNS-83-03680,
`Ripon College Faculty Development Funds, and the Continental Coffee Products
`Company (a wholly owned subsidiary of Quaker Oats), The excavations were
`carried out under Credencial 038-83-DCIRBM,issued by the Instituto Nacional de
`Cultura of Peru, We thank A, A. Hunter (Missouri) whoidentified the squash seeds
`and A. Price, J. Atteberry, and L. Haubrich whohelped in sorting andtallying data.
`Additional aid in processing the subsistence remains was given by N. Salazar and
`M. C. Rodriguez de Sandweiss in Peru. L. Salazar-Burger, assistantfield director,
`was essential to the project. The Centro de Investigaciones de Zonas Ardias was our
`base ofoperations and analysis and we thank F, A. Engel and M. Vallejos and many
`other Peruvian collegues for support.
`
`
`
`Chemistry and Biology of the Immunophilins
`and Their Immunosuppressive Ligands
`
`STUART L. SCHREIBER
`
`Cyclosporin A, FK506, and rapamycin are inhibitors of
`specific signal transduction pathways that lead to T lym-
`phocyteactivation. These immunosuppressive agents bind
`with highaffinity to cytoplasmic receptors termed immu-
`nophilins (immunosuppressant binding proteins), Studies
`in this area have focused on the structural basis for the
`molecular recognition of immunosuppressants by immu-
`nophilins and the biological consequences of their inter-
`actions. Defining the biological roles of this emerging
`family of receptors and their ligands may illuminate the
`process ofprotein trafficking in cells and the mechanisms
`of signal transmission through the cytoplasm.
`
`ESEARCH DURING THE PAST DECADE HAS CONTRIBUTED
`
`significantly to our knowledge of T lymphocyte function.
`The identification and functional analysis of T cell surface
`receptors (7) and nuclear transcription factors (2) have made these
`components of the signal transduction apparatus among the best
`understood in biology. This understanding is largely due to the use
`ofprobe reagents, such as monoclonal antibodies and radiolabeled
`nucleic acids, that have been developed for the study of surface and
`nuclear phenomena, respectively. However, the mechanisms for the
`transduction of signals through the cytoplasm, the “black box” of
`the signal transduction pathway, remain mysterious.
`A family of natural products has emerged as probe reagents for
`cytoplasmic signaling mechanisms in the T lymphocyte. These small
`
`The authoris a professor of Chemistry, Harvard University, Cambridge, MA 02138.
`18 JANUARY 1991
`
`molecules are immunosuppressants that appear to exert their inhib-
`itory actions distal to early membrane-associated events and proxi-
`malto nuclear processes. Studies on a family of immunosuppressant
`binding proteins, the immunophilins, have attempted to identify the
`structural requirements for high-affinity interactions between immu-
`nophilins and their immunosuppressive ligands and the biological
`consequences of the formation of immunophilin-ligand complexes,
`Although there is much to explore in this avenue ofresearch, some
`general principles associated with the intermediary events of signal
`processing are emerging.
`
`The Immunosuppressants
`Cyclosporin A (CsA), aninhibitor ofT cell activation,is currently
`the favored therapeutic agent for prevention ofgraft rejection after
`organ and bone marrowtransplantation, and it has been credited
`with initiating a revolution in clinical transplantation (3-5), The
`recently discovered compound FK506 inhibits T cell activation by
`mechanismsthatare similar to those of CsA, but FK506 is 10 to 100
`times as potent (6). FK506 has performed remarkably well in initial
`humanclinical transplantation trials (7, 8), despite reports of toxic
`effects in animals (6). Rapamycin inhibits T cell activation at
`concentrations comparable to those of the structurally related
`FK506, yet with mechanismsthatare strikingly different from those
`mediated by FK506, and thus CsA (9), Only CsA, FK506, and
`rapamycin have been used for the identification of members of the
`immunophilin class. A nonnatural ligand, 506BD (10), and analogs
`of CsA (11-13) have also provided insights into the inhibitory
`mechanisms of immunosuppressants. Many recently discovered
`immunosuppressive agents (14) with undefined mechanisms, suchas
`ARTICLES
`283
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`discodermolide (15) and deoxyspergualin (16), promise to reveal
`new facets of cytoplasmic signaling mechanisms (17) (Fig. 1).
`
`The Immunophilins
`The predominant CsA-binding protein in T lymphocytes is the
`soluble, cytosolic receptor cyclophilin (18, 79), Cyclophilin is an
`abundant and ubiquitous protein that is found in both prokaryotic
`and eukaryotic organisms. The major isoform of human cyclophilin
`has a mass of 17,737 daltons and anisoelectric point (pI) of 9.3.
`Two groups have independently reported that cyclophilin is identi-
`cal to peptidyl-prolyl isomerase (20, 21), an enzyme thatcatalyzes
`the interconversion of the cis- and trans-rotamers of the peptidyl-
`prolyl amide bond of peptide and protein substrates, and this
`rotamase activity is potently inhibited by CsA.
`Shortly after this discovery,
`the predominant FK506-binding
`protein in calf thymus, human spleen, and the T cell line Jurkat,
`termed FKBP, was isolated and characterized in two laboratories
`(22, 23). Like cyclophilin, FKBP was shown to have rotamase
`activity toward a peptide substrate. FK506 inhibits the rotamase
`activity of FKBP, but not of cyclophilin; likewise, CsA does not
`inhibit the rotamase activity of FKBP, The cloning (24, 25) and
`overexpression (24) of human recombinant FKBP and the cloning
`of an FKBP from Neurospora crassa (26) revealed that, despite their
`common enzymatic properties, FKBP and cyclophilin have dissim-
`ilar sequences. Human FKBPhas a mass of 11,819 daltons and,like
`cyclophilin,is a basic (pI = 8.9) (22, 24), cytosolic protein (27). A
`prokaryotic organism, Neisseria meningitidis, was found to have an
`open reading frame that encodes an FKBP-like protein (24), More
`recently, FKBP was shown to be the predominant rapamycin-
`binding protein in yeast, calf thymus, and human T cells (Jurkat)
`(28). Rapamycin (dissociation constant Ky = 0.2 nM) has an even
`higheraffinity for FRBP than does FK506 (Ky = 0.4 nM), and is
`also a potent
`inhibitor of FKBP’s rotamase activity (inhibition
`
`
`
`506BD Rapamycin
`
`ge o
`
`
`Mo
`4
`
`
`
`oco
`
`Mo Mo Ma
`
`OMe
`
`“OH
`
`
`~doyAdmenanerson,
`
`On
`Goa
`
`
`
`Mo
`OH OCONH,
`
`Hi
`
`o
`
`Discodermolide
`
`Deoxyspergualin
`
`Fig. 1. Probe reagents ofintracellular signaling pathways. (A) Recently
`investigated immunophilin ligands.
`(B) Immunosuppressive agents with
`unknown mechanisms of T cell inhibition. (Me, methyl.)
`284
`
`constant K; = 0.2 nM) (29).
`Although cyclophilin and FKBP are the only well-characterized
`immunophilins, other membersofthis family are knownto exist and
`are currently being investigated. For example, a CsA-binding phos-
`phoprotein ofrelative molecular mass (M,) 45,000 has been detect-
`ed in Jurkat cells (30), and phosphoproteins of M, 60,000 and
`80,000 from this samecell line bind to both FK506 and rapamycin
`(28). The ninaA gene of Drosophila (31, 32) and a second cyclo-
`philin-related gene in Saccharomyces cerevisiae (33) encode proteins
`that show high homology to cyclophilin. Several low molecular
`weight, basic proteins that are retained on CsA, FK506, or rapa-
`mycin affinity matrices have also been noted (22, 28). Partial
`sequence determination of FK506- and rapamycin-binding immu-
`nophilins of M, 30,000 and M, 13,000 has revealed that these
`molecules,
`together with FKBP, are members of a previously
`unknownfamily of immunophilins (34). Questions concerning the
`biological relevance, the rotamase activity, and theaffinity to the
`cognate ligands of these low-abundance immunophilins should soon
`be answered.
`Although the exact cellular concentrations of FKBP and cyclo-
`philin are not known, both are abundant, Saturation binding in the
`cytosol of Jurkat cells was reported to occur at >5 nM _ditritio-
`FK506 (27). As FKBP is the predominant cytosolic receptor for
`drug, this measurementis largely accounted for by FKBP, and thus
`the cytoplasmic concentration of FKBP may approach 5 nM. The
`high-affinity FKBP ligands FK506 and rapamycin, however, inhibit
`T cell proliferation at subnanomolar concentrations (median inhibi-
`tion concentration IC;, ~0.5 nM) (29, 35). Therefore,inhibition of
`the rotamase activity of FKBPis very likely an insufficient require-
`ment for mediating the actions of these drugs in T lymphocytes,
`because only a small fraction of the enzyme wouldbe inhibited at
`effective drug concentrations. This point has been confirmed by
`mechanistic studies of FK506 and rapamycin (see below); likewise,
`investigations ofCsA analogs support a similar conclusion regarding
`the rotamaseactivity of cyclophilin (12).
`
`Molecular Recognition by the Immunophilins
`The rotamase activity of these immunophilins and the ability of
`their immunosuppressive ligands to act as
`rotamase inhibitors
`provide an opportunity for exploration ofthe molecular basis for the
`high-affinity interactions that exist between them. Initial mechanistic
`studies of cyclophilin led to the suggestion that catalysis ofthe
`interconversion ofcis- and frans-rotamers of a peptide substrate is
`achieved by the formation of a covalent bond to the carbonyl ofthe
`peptidyl-prolyl amide with a cysteine-derived thiol (36). Loss of
`amide resonance would be expected to lower the activation barrier
`to rotation about the amide C-N bond.Site-directed mutagenesis of
`human recombinantcyclophilin allowed the systematic replacement
`ofall four cysteine residues in cyclophilin with alanine, Becauseall
`four mutants enzymes were fully active in the rotamase andbinding
`assays, cystcine was ruled out as a participating residue in catalysis
`(37).
`Additional mechanistic studies with both cyclophilin (38) and
`FKBP (39) strongly suggest that these enzymes catalyze rotamer
`interconversion by noncovalent stabilization of the twisted amide
`transition state for the noncatalyzed isomerization. The amide
`functionality exhibits a strong preference for a planar geometry,
`wherein the nitrogen lone pair is in conjugation with the carbonyl
`ar-cloud. The energy cost of the twisted amidestructure (Fig. 2A)is
`15 to 20keal (40). The structural basis for cyclophilin and FKBP’s
`ability to stabilize this transition-state structure must await further
`structural analyses of rotamase-peptide (or inhibitor) complexes.
`SCIENCE, VOL. 251
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`
`Fig. 2. (A) Modelofthe transition state structure ofa twisted peptidyl-prolyl
`amide bond that is stabilized by the rotamase enzymes cyclophilin and
`FKBP. (B) Substructure of FK506 and (C) CsA (both from x-ray) that is
`proposed to mimic the twisted amide bond of a peptide substrate. (D)
`Substructure of FK506 (R = OMe) and rapamycin (R = H) proposed to
`mimic a twisted leucyl-prolyl amide bond of a peptide substrate.
`(E)
`Leucyl-prolyl fragment indicating structural similarities to immunosuppres-
`sant substructures,
`
`However, the unusual structure of the immunophilin ligands and
`preliminary structural
`investigations of the immunophilin-ligand
`complex suggesta basis for their rotamase inhibitory properties. The
`total synthesis of a '$C-labeled FK506 (41) provided a reagent to
`carry out °C nuclear magnetic resonance (NMR) studies of the
`FK506-FKBP complex (42). It was suggested that
`the ketone
`carbonyl adjacent to the homoprolyl amide bond of FK506 (Fig.
`28) and rapamycin is a mimic of the amide carbonyl of a peptide
`substrate, Thus, FK506 and rapamycin are transition-state analogs
`in that their ground-state geometry is similar to the transition-state
`structure of a peptide substrate (Fig. 2, A and B). Also, the side
`chain of the unusual amino acid N-methyl-butenylthreonine
`(MeBmt) of CsA, which is known to be essential for high-affinity
`binding of CsA to cyclophilin (17, 12), has structural similarity (Fig.
`2C)to the aforementionedtransition-state structure (Fig. 2A). This
`side chain maybe a different type ofsurrogate for the twisted amide
`structure. In this regard, the a-branched hydroxyethylene substruc-
`ture of CsA is reminiscent of the hydroxyethylene amide isostere
`found in aspartyl protease inhibitors such as pepstatin.
`The analogy of the «-keto-homoproly! grouping in FK506 and
`rapamycin to a twisted-amide bond of a peptide substrate was
`extended (39). A substrate containing a leucyl-prolyl dipeptide was
`found to be optimal for FKBP (39, 43). The structuralsimilarities of
`FK506 and rapamycin to a twisted leucyl-amide bond (Fig. 2, D
`and E) suggest these agents may be transition-state analogs of a
`leucyl-(twisted amide)-prolyl peptide substrate for FKBP.
`
`The Biological Function of Immunophilins
`The complex series of events that comprises the T cell activation
`cascade transpires over several days (2). CsA, FK506, and rapamycin
`act within the first hours of the process (Fig. 3). Stimulation of the
`T cell receptor (TCR) by foreign antigen presented by a major
`histocompatibility (MHC) molecule on the surface of an antigen-
`18 JANUARY 199]
`
`presentingcell results in the activation of a TCRsignal transmission
`pathway. The signal is transduced through the cytoplasm by an
`unknown mechanismandresults in the activation ofspecific nuclear
`transcription factors, such as nuclear factor of activated T cells
`(NF-AT). These nuclear factors help to regulate the transcription of
`T cell activation genes, such as the gene of the lymphokine inter-
`leukin-2 (IL-2). Translation of the resultant message is followed by
`secretion of IL-2. CsA and FK506 are potent inhibitors of the
`TCR-mediated signal transduction pathway, as evidenced by their
`ability to inhibit the transcription of early T cell activation genes
`(44). CsA (45) and FK506 (29, 46), but not rapamycin, inhibit the
`binding of NF-AT to the IL-2 enhancer and inhibit transcriptional
`activation by NF-AT. CsA and FK506 also inhibit transcription
`mediated by AP-3 and Oct-1, and partially inhibit transcription
`mediated by NF-«B (45, 46). Anotherillustration involves the use of
`T cell hybridomas that undergo a suicidal event called apoptosis
`after stimulation of the TCR-CD3 complex. CsA and FK506, but
`not rapamycin, are potent inhibitors of apoptosis induced by an
`antibody to the TCR-CD3 complex (29).
`T cell activation involves not only IL-2 secretion but also expres-
`sion of the lymphokine receptor IL-2R on the surface ofthe cell.
`After the binding of IL-2 to IL-2R, a lymphokine receptor (LKR)
`signal transmission pathwayis activated. Transductionofthis signal
`again proceeds by an unknown mechanism through the cytoplasm
`and into the nucleus, where a different set of genesis transcribed.
`Whereas rapamycin, despite its structural similarity to FK506, has
`no effect on the production of IL-2, it potently inhibits the response
`of the T cell to IL-2 (29, 35, 47). Rapamycin thus appears to inhibit
`a later LKR-associated signaling pathway (Fig. 3). Because both
`rapamycin and FK506are potentinhibitors of the rotamaseactivity
`of FKBP and inhibit distinct signaling pathways,
`these results
`support the suggestion that the inhibition of rotamase activity of
`FKBPis an insufficient requirement for mediating the actions of
`FK506 and rapamycin (10, 29).
`In addition to their ability to inhibit different T cell activation
`events, rapamycin and FK506, but not CsA, have been shownto be
`mutually inhibitory in a variety of functional assays (29, 47). These
`results suggest a role for cither a single immunophilin or separate
`immunophilins that share a commonreceptorsite in mediating the
`actions of FK506 and rapamycin, Furthermore,
`rapamycin can
`distinguish the biological actions of FK506 and CsA, because it has
`no effect on the actions of CsA.
`The mutual inhibition of FK506 and rapamycin was shown to be
`subject to a buffering action by FKBP (29). A concentration 10 to
`100 times the effective drug concentration (IC5, ~ 0.5 nM) of
`
`(1) TGR signal
`
`(2)LKR signal ez Co
`
`
`
`
`hes, é
`call differentiation
`cell proliferation
`
`Fig. 3. Early events ofthe T cell activation cascade andthesites of inhibitory
`action by CsA, FK506, and rapamycin.
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`Immune ra|+CUG EKBP (—_> protein translocation
`
`FKBP/drug
`Foldase,
`cormyiene
`Ma
`
`
`@ =e
`elc.
`modulation
`B
`o
`inding
`it w
`domain
`‘~
`antagonist (_)
`
`oO FKBP/antagonist
`complex
`
`Fig. 4. Schematicillustrating the relative abundance ofreceptor and ligands.
`The abundant FKBP, which may have a cellular function as a foldase,
`is
`converted to an inhibitory complex on binding of the drug and buffers the
`actions of the antagonist.
`
`ee.
`
`me
`
`““OH
`
`
`
`Se MaMeMeA Sou
`
`
`
`
`
`either agent is required for inhibition ofthe actions of the other(29,
`47). Thus,
`the abundant (~5 nM) uncomplexed immunophilin
`sequesters the antagonist. Only after the excess binding sites are
`occupied does the concentration of the antagonizing agent
`rise
`sufficiently to displace the drug effectively from its biological
`
`
`remove
`receptor. These findings also implicate the immunophilin-drug com-
`effector
`element
`plex as the inhibitor ofTcell activation. Althougha role for FKBP as
`the mediator ofthe biological actions of rapamycin and FK506 has
`not been shown,the buffer effect of FKBPevident in the studies of
`reciprocal inhibition should also be operative with competing cellular
`receptors for these drugs. The low-abundance immunophilins must
`overcome the high abundance of FKBP andits high affinity for drug
`in order to competeeffectively for binding (Fig. 4).
`Invoking the immunophilin-drug complex as the biological effec-
`tor addresses the issue of how the ubiquitous cyclophilin and FKBP
`could be involved in T cell activation. One possibility is that these
`proteins have a more general function, perhaps assisting in protein
`folding in vivo byacting as foldases. Only when the immunophilin
`combines with its immunosuppressive ligand doesit inhibit 'T cell
`activation. The cellular immunophilin receptor (possibly FKBP),
`bound to either FK506 or rapamycin, may interact with different
`molecules in distinct pathways ofT cell activation. Accordingto this
`hypothesis, the specificity of the factors associated with different
`signaling pathways is determined by the precise geometry of the
`immunophilin-drug complex. Evidence has been presented (48) that
`the cyclophilin-CsA complex, and not CsA,is the agent responsible
`for the toxic actions of CsA in two lower cukaryotes. CsA-sensitive
`strains of N. crassa and S. cerevisiae were grownin the presence of
`CsA. Analysis of the CsA-resistant mutant strains that resulted
`revealed that either they no longer produced cyclophilin or, if they
`did, the cyclophilin of the mutantstrains did not bind CsA (48).
`The common biological receptor site implied by the mutual
`inhibition of FK506 and rapamycin suggests that the immunophilin
`may present multiple ligands to cytoplasmic components ofsignal
`transmission pathways. The ability of a single immunophilin to
`present two immunosuppressive ligandsto effectors associated with
`two distinct pathwaysraises the possibility thar immunophilins may
`function as general presenting molecules, by analogy to the way that
`MHCmolecules presenta large numberofpeptides to the polymor-
`phic TCRs. Ifendogenous immunophilin ligandsexist that function
`similarly to the immunosuppressive natural products,
`then the
`immunesystem may have used the molecular recognition associated
`with rotamase catalysis for
`the purpose of modulating T cell
`activation.
`In the case of FK506 and rapamycin, the leucyl-(twisted amide)-
`prolyl peptidomimetic fragment shared by these drugs constitutes
`the structural elementlargely responsible for binding to FKBP. This
`common immunophilin binding domain is then fused to distinct
`effector elements that, after presentation by the immunophilin,
`determine the signaling pathway with which the drug will interfere
`(Fig. 5, A and B). This view of FK506 and rapamycin as dual
`domain agents was tested with an FKBP ligand designed to contain
`286
`
`Future Prospects
`The presence of cyclophilin and FKBP in many organisms
`suggests that these enzymes may have some generalcellular func-
`tion. The recent discoveries of proteins that assist in protein folding,
`unfolding, and translocation in vivo provide precedent for a similar
`function for rotamase enzymes (49). A role for an Escherichia coli
`cyclophilin in the secretory pathway was suggested after the discov-
`ery that
`it was localized in the periplasm (50). Similarly,
`the
`demonstration that an N, crassa FKBPcatalyzed protein folding and
`the identification of mitochondrial forms of N. crassa cyclophilin
`and FKBPled to the suggestion that these immunophilinsassist in
`the refolding ofproteins that have traversed a biological membrane
`(26). The identification and characterization of new immunophilins
`will increase our understanding ofthese molecules. The structures of
`immunophilins and their drug complexes may prove illuminating, in
`regard to both enzyme mechanism andcell signaling inhibition.
`Many questions remain unanswered concerning the mechanisms
`SCIENCE,VOL, 251
`
`Fig. 5. Domainal analyses of FKBP ligands. (A) FK506 and (B) rapamycin
`binding domain and effector elements (shaded). (C) Structure of FK506
`(x-ray) with enolate spacer drawntoillustrate scaffolding effect. (D) Removal
`of the outer loop of structure (C) results in 506BD,a high-affinity (K, = 5
`nM) ligand to FKBP.
`
`the putative FKBP-binding domain of FK506 and rapamycinin the
`conformation found in the solid state of FK506 (Fig. 5C) (10). The
`resultant molecule, 506BD, was found to bind with highaffinity (K,
`= 20 nM)and to inhibit the rotamaseactivity (K, = 5 nM) of FKBP
`potently (Fig. 5D), Because 506BD lacks the putative effector
`elements of either FK506 or rapamycin,
`it was not expected to
`inhibit either the TCR or LKRsignaling pathways associated with
`T cell activation. Indeed, 506BD does not inhibit T cell activation
`by either mechanism, even at high concentrations. However, this
`immunophilin ligand inhibits the actions of both FK506 and
`rapamycin at concentrations that would be anticipated given the
`relative affinity of these agents to F