Scientific Side Trips: Six Excursions from the Beaten Path
2012; Elsevier BV; Volume: 287; Issue: 27 Linguagem: Inglês
10.1074/jbc.x112.381681
ISSN1083-351X
AutoresMichael S. Brown, Joseph L. Goldstein,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoOur 40-year research partnership began on a sunny June day in 1966 at the Massachusetts General Hospital, where we met with twelve other nervous neophytes to begin our internships in internal medicine. Within a few days, we developed the friendship and mutual respect that were to sustain our collaborative effort over four decades. Our backgrounds were quite different. Joe grew up in small-town South Carolina and graduated from the young University of Texas Southwestern Medical School in Dallas, where he came under the spell of Donald W. Seldin, chairman of medicine and intellectual father to generations of physician-scientists. In stark contrast, Mike grew up in big-city Philadelphia and graduated from the nation's oldest medical school, the University of Pennsylvania School of Medicine. We were drawn together by a shared fascination with clinical medicine and medical science and a desire to one day make discoveries of significance to both. In 1968, after two years of residency, we were both accepted for scientific training at the National Institutes of Health (NIH), where Joe worked in the laboratory of Marshall Nirenberg, who was soon to receive a Nobel Prize for solving the genetic code. Mike worked with Earl Stadtman, the biochemist's biochemist who later received the United States National Medal of Science. We also had clinical duties. Joe was assigned to care for a pair of siblings (ages 6 and 8) who were suffering repeated myocardial infarctions due to massively elevated levels of low-density lipoprotein (LDL) cholesterol in their blood. The diagnosis was homozygous familial hypercholesterolemia (FH), a rare form of a common genetic disease, the pathogenesis of which was unknown. The two of us discussed these children intensely, and we resolved some day to unlock the genetic secret behind this striking illness. In 1970, after two years at the NIH, Joe moved to Seattle to learn medical genetics under the skillful tutelage of Arno Motulsky. There, Joe spearheaded a classic study of the common Mendelian causes of hyperlipidemia in survivors of myocardial infarction. Mike remained one more year at the NIH. In 1971, he moved to the University of Texas Southwestern Medical Center, where he completed a fellowship in gastroenterology and began to study the properties of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-controlling enzyme of cholesterol synthesis that was later shown by Akira Endo to be the target of the statin drugs (Ref. 1Endo A. Kuroda M. Tsujita Y. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum.J. Antibiot. 1976; 29: 1346-1348Crossref PubMed Google Scholar; see Ref. 2Brown M.S. Goldstein J.L. A tribute to Akira Endo, discoverer of a "Penicillin" for cholesterol.Atherosclerosis Suppl. 2004; 5: 13-16Abstract Full Text Full Text PDF PubMed Google Scholar). Mike's move to Dallas was strongly advocated by Joe, who enlisted the aid of Seldin. Mike was willing to try this young medical school. Amazingly, his wife, Alice, a born-again Yankee, agreed to the move, but only under the condition that they would stay for one or two years at most. That was four decades ago, and the couple is still happily in residence, having raised two Texan daughters. In 1972, Joe returned to Dallas, and we began our collaborative studies of FH. This work led to the discovery of the LDL receptor, the process of receptor-mediated endocytosis, and the mechanism by which LDL receptors control the level of cholesterol in blood. All of this early work (1972–1985) has been reviewed elsewhere, most notably in our Nobel lecture, which was published in Science in 1986 (3Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Google Scholar). It has also been detailed in more recent historical reviews (4Goldstein J.L. Brown M.S. History of discovery: the LDL receptor.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 431-438Crossref PubMed Scopus (0) Google Scholar, 5Brown M.S. Goldstein J.L. In memoriam: Richard G. W. Anderson (1940–2011) and the birth of receptor-mediated endocytosis.J. Cell Biol. 2011; 193: 601-603Crossref PubMed Scopus (2) Google Scholar). In 1993, our study of the regulation of LDL receptors led to the discovery of sterol regulatory-element binding proteins (SREBPs), transcription factors that induce lipid synthesis and uptake in animal cells (6Wang X. Briggs M.R. Hua X. Yokoyama C. Goldstein J.L. Brown M.S. Nuclear protein that binds sterol regulatory element of LDL receptor promoter. II. Purification and characterization.J. Biol. Chem. 1993; 268: 14497-14504Abstract Full Text PDF PubMed Google Scholar, 7Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. SREBP-1, a basic helix-loop-helix leucine zipper protein that controls transcription of the LDL receptor gene.Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (744) Google Scholar, 8Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis.Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (814) Google Scholar, 9Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 11603-11607Crossref PubMed Google Scholar). The crucial purification was performed by two postdoctoral fellows, Xiaodong Wang and Michael Briggs, and the cDNA cloning was accomplished by Chieko Yokoyama, a postdoctoral fellow, and Xianxin Hua, a graduate student. The novel feature of SREBPs is that they are synthesized as intrinsic membrane proteins of the endoplasmic reticulum (ER), and they must be transported to the Golgi complex, where they are cleaved to send active fragments to the nucleus. We described this transport process and its feedback regulation in detail and named it regulated intramembrane proteolysis (RIP). Like receptor-mediated endocytosis, RIP turned out to be a fundamental biologic mechanism that is used in more than forty other regulatory systems, including the proteolytic processing of the developmental protein Notch, the stress protein ATF6, and the amyloid precursor protein, which is cleaved by RIP to form the pathologic amyloid-β peptide (10Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans.Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Google Scholar, 11Lal M. Caplan M. Regulated intramembrane proteolysis: signaling pathways and biological functions.Physiology. 2011; 26: 34-44Crossref PubMed Scopus (62) Google Scholar). Earlier review articles detail the combined genetic and biochemical approaches that we used to delineate the transport proteins and proteases responsible for RIP of SREBPs and to show how these cleaved transcription factors activate the complete program of cholesterol and fatty acid synthesis in the liver (12Goldstein J.L. Rawson R.B. Brown M.S. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis.Arch. Biochem. Biophys. 2002; 397: 139-148Crossref PubMed Scopus (185) Google Scholar, 13Horton J.D. Goldstein J.L. Brown M.S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver.J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Google Scholar, 14Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1095) Google Scholar, 15Brown M.S. Goldstein J.L. Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.J. Lipid Res. 2009; 50: S15-S27Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In this Reflections article, we will not discuss LDL receptors, SREBPs, or cholesterol homeostasis. Rather, we focus on six side projects that we pursued over the years, each affording an adventurous diversion from the central core of our research and each teaching us a principle useful to science and medicine. In describing each project, we credit the students and postdoctoral fellows who were most responsible. However, we do not mean to slight the many others who made important, even crucial, observations. We value all of them. Similarly, in citing work from other laboratories, we focus on the papers that stimulated us or expanded on our work. This is a very personal list, and we apologize in advance to the many unnamed scientists who made substantial contributions to each field. This article represents a personal reflection and not a comprehensive review. Our discovery of scavenger receptors began with a pathogenic paradox. Patients with homozygous FH lack LDL receptors; therefore, their cells cannot take up LDL, yet the same patients accumulate massive amounts of LDL-derived cholesterol in scavenger cells of the body, primarily macrophages. Moreover, even in subjects with normal LDL receptors, it is impossible to overload macrophages with cholesterol by incubation with LDL. When macrophages, or any other cells, begin to accumulate cholesterol, LDL receptors are reduced by feedback inhibition of SREBP processing, yet LDL cholesterol accumulates to high levels in macrophages within atherosclerotic plaques, converting them into foam cells. Clearly, macrophages must have an alternate, non-suppressible pathway to take up LDL. In 1978, together with Y. K. Ho and Sandip Basu, our first two postdoctoral fellows, we isolated macrophages from mouse peritoneal fluid and incubated them with native LDL (16Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Binding site on macrophages that mediates uptake and degradation of acetylated low-density lipoprotein, producing massive cholesterol deposition.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 333-337Crossref PubMed Google Scholar). As expected, the cells did not accumulate excess cholesterol because their LDL receptors became down-regulated (Fig. 1A). We had earlier shown that acetylation of lysine residues on LDL destroys its ability to bind to LDL receptors, and we wondered whether the acetyl-LDL would have acquired the ability to be taken up by macrophages. To our delight, this was the case (Fig. 1B). Macrophages expressed a high-affinity receptor that bound and internalized acetyl-LDL without any down-regulation. The excess cholesterol was esterified and stored in the cytosol as cholesteryl esters, whose concentration was increased by 40-fold in the cells, converting them to classic foam cells. Remarkably, the macrophage receptor was not specific for acetyl-LDL. Other negatively charged macromolecules, including maleyl-LDL, maleyl-albumin, sulfated polysaccharides (such as fucoidan and dextran sulfate), and polynucleotides (such as poly(I) and poly(G)) competed for acetyl-LDL uptake. There was some selectivity, however. Certain negatively charged polymers (such as poly(C), poly(A), and poly(d-glutamic acid)) did not compete (17Brown M.S. Basu S.K. Falck J.R. Ho Y.K. Goldstein J.L. The scavenger cell pathway for lipoprotein degradation: specificity of the binding site that mediates the uptake of negatively charged LDL by macrophages.J. Supramol. Struct. 1980; 13: 67-81Crossref PubMed Google Scholar). The acetyl-LDL receptor was expressed only on scavenger cells, including guinea pig Kupffer cells and human activated monocytes. We speculated that this receptor is designed to scavenge certain denatured proteins and other macromolecules from the circulation and that it might be responsible for cholesteryl ester accumulation in human atherosclerotic plaques (18Brown M.S. Goldstein J.L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis.Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). Using the scavenger receptor to mediate cholesterol overload, we next defined the macrophage cholesteryl ester cycle (19Brown M.S. Ho Y.K. Goldstein J.L. The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters.J. Biol. Chem. 1980; 255: 9344-9352Abstract Full Text PDF PubMed Google Scholar). After uptake, cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and the free cholesterol is re-esterified in the ER for storage as cholesteryl ester droplets. The key finding was that the cells required an external cholesterol acceptor to excrete the stored cholesterol from the cell. When we added an external acceptor, such as high-density lipoprotein (HDL), the stored cholesteryl esters were hydrolyzed by a cytosolic neutral lipase, and the cholesterol was released to the acceptor. HDL was not the only acceptor in blood. Indeed, native LDL could serve as an acceptor, and so could other plasma proteins, such as thyroglobulin, but surprisingly not albumin. The most potent acceptors were membranes prepared from red blood cells, suggesting that red blood cells may be active participants in the shuttling of cholesterol between organs (20Ho Y.K. Brown M.S. Goldstein J.L. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high-density lipoprotein and other agents.J. Lipid Res. 1980; 21: 391-398Abstract Full Text PDF PubMed Google Scholar). To our knowledge, this hypothesis has not been tested, but it may be important in the process that is now known as reverse cholesterol transport. We also observed that addition of acetyl-LDL to macrophages stimulated the cells to produce apolipoprotein E (apoE), which can target lipoproteins for uptake by the liver (21Basu S.K. Brown M.S. Ho Y.K. Havel R.J. Goldstein J.L. Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E.Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 7545-7549Crossref PubMed Google Scholar). This finding presaged the later observations of Peter Tontonoz, Peter Edwards, and David Mangelsdorf, who showed that cholesterol loading of macrophages leads to activation of the nuclear hormone liver X receptor. This receptor activates transcription of a variety of genes (including those for apoE and ABCA1) that are designed to release stored cholesterol from macrophages (22Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXRα.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 12097-12102Crossref PubMed Scopus (784) Google Scholar, 23Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 507-512Crossref PubMed Google Scholar). We have one regret about the macrophage studies, and it involves missing oxidized LDL. In thinking about a possible physiologic ligand for the scavenger receptor, we considered that LDL might undergo oxidation, and some of the oxidation products may have attached to lysine residues, giving the LDL a negative charge. Accordingly, we put human LDL in a dialysis bag immersed in a flask filled with buffer. We bubbled oxygen through the solution overnight, and the next day, we tested the LDL for uptake by macrophages. The result was negative. We had neglected to consider that our buffer contained EDTA, which binds the trace metals that are needed for the lipid oxidation reaction. Daniel Steinberg and colleagues did the right experiment and found that oxidized LDL did indeed bind to the macrophage receptor (24Henriksen T. Mahoney E.M. Steinberg D. Enhanced macrophage degradation of low-density lipoprotein previously incubated with cultured endothelial cells: recognition by receptors for acetylated low-density lipoproteins.Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 6499-6503Crossref PubMed Google Scholar, 25Steinberg D. Chance and serendipity in science: two examples from my own career.J. Biol. Chem. 2011; 286: 37895-37904Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), and Alan Fogelman and colleagues showed that oxidation of unsaturated fatty acids in LDL generates malondialdehyde, which forms Schiff bases with lysines, removing the positive charge and mimicking acetylation (26Fogelman A.M. Shechter I. Seager J. Hokom M. Child J.S. Edwards P.A. Malondialdehyde alteration of low-density lipoproteins leads to cholesteryl ester accumulation in human monocyte-macrophages.Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 2214-2218Crossref PubMed Google Scholar). Although definitive proof is still lacking, the accumulated evidence suggests that LDL becomes oxidized in atherosclerotic plaques, and this causes it to bind to scavenger receptors, creating the foam cells that are the hallmark of this disease (27Steinberg D. Witztum J.L. History of discovery: oxidized low-density lipoprotein and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2311-2316Crossref PubMed Scopus (0) Google Scholar). Antibodies to oxidized LDL may prove to be useful in blocking macrophage cholesterol accumulation and preventing myocardial infarctions (28Binder C.J. Hartvigsen K. Witztum J.L. Promise of immune modulation to inhibit atherogenesis.J. Am. Coll. Cardiol. 2007; 50: 547-550Crossref PubMed Scopus (24) Google Scholar). In 1990, the acetyl-LDL receptor was purified and its cDNA was cloned by our former postdoctoral fellow Monty Krieger and his postdoctoral fellows at the Massachusetts Institute of Technology (29Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Type I macrophage scavenger receptor contains α-helical and collagen-like coiled coils.Nature. 1990; 343: 531-535Crossref PubMed Scopus (814) Google Scholar). Now called SR-AI (for scavenger receptor class A, type I), the original scavenger receptor has been joined by a family of twelve other structurally diverse membrane receptors, all of which take up acetyl-LDL and/or oxidized LDL. They also bind and internalize a wide range of other ligands, including lipid molecules displayed on the surface of bacterial pathogens and modified self-molecules displayed on apoptotic cells. The extensive literature on the roles of scavenger receptors in innate immunity, microbial pathogenesis, and various pathologic processes (in addition to atherosclerosis) has been described nicely in a series of review articles by Siamon Gordon and colleagues (30Areschoug T. Gordon S. Scavenger receptors: role in innate immunity and microbial pathogenesis.Cell. Microbiol. 2009; 11: 1160-1169Crossref PubMed Scopus (194) Google Scholar, 31Greaves D.R. Gordon S. The macrophage scavenger receptor at 30 years of age: current knowledge and future challenges.J. Lipid Res. 2009; 50: S282-S286Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). We became interested in protein prenylation when we read the papers of John Glomset and co-workers, who showed that certain proteins in animal cells possess hydrophobic prenyl groups, 15-carbon farnesyl or 20-carbon geranylgeranyl attached to cysteines in thioether linkage (32Schmidt R.A. Schneider C.J. Glomset J.A. Evidence for post-translational incorporation of a product of mevalonic acid into Swiss 3T3 cell proteins.J. Biol. Chem. 1984; 259: 10175-10180Abstract Full Text PDF PubMed Google Scholar). Prominent farnesylated proteins include Ras proteins, whose activating mutations are among the most common causes of cancer. Remarkably, Glomset's findings were made possible by Akira Endo's discovery of HMG-CoA reductase inhibitors (1Endo A. Kuroda M. Tsujita Y. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum.J. Antibiot. 1976; 29: 1346-1348Crossref PubMed Google Scholar). Thus, a discovery aimed toward heart disease increased our fundamental knowledge of cancer. So much for categorical NIH institutes! All prenyl groups are derived from mevalonate, which is produced by HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway (33Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Google Scholar). Glomset's discovery was triggered by his observation that blocking HMG-CoA reductase with a statin precluded cells from entering the S phase of the cell cycle. Adding cholesterol did not reverse this deficiency, suggesting that the cells needed another product derived from mevalonate. While blocking production of endogenous mevalonate with a statin, Glomset labeled the cells with [3H]mevalonate and found incorporation into several proteins (32Schmidt R.A. Schneider C.J. Glomset J.A. Evidence for post-translational incorporation of a product of mevalonic acid into Swiss 3T3 cell proteins.J. Biol. Chem. 1984; 259: 10175-10180Abstract Full Text PDF PubMed Google Scholar). Work in yeast and animal cells soon showed that one set of prenylated proteins always terminates in the amino acid sequence CAAX, where A stands for aliphatic, and X can be one of several residues (34Schafer W.R. Trueblood C.E. Yang C.C. Mayer M.P. Rosenberg S. Poulter C.D. Kim S.H. Rine J. Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the Ras protein.Science. 1990; 249: 1133-1139Crossref PubMed Google Scholar, 35Lowy D.R. Willumsen B.M. New clue to Ras lipid glue.Nature. 1989; 341: 384-385Crossref PubMed Google Scholar). The X residue determines whether a protein will be modified with a farnesyl or a geranylgeranyl. Oncogenic Ras proteins are farnesylated, whereas structural proteins like Rho and Rac are geranylgeranylated (36Glomset J.A. Gelb M.H. Farnsworth C.C. The prenylation of proteins.Curr. Opin. Lipidol. 1991; 2: 118-124Crossref Scopus (0) Google Scholar). After prenylation, the proteins are cleaved by a protease, so the prenylated cysteine becomes the COOH-terminal amino acid, and its free COOH group is then methylated, removing all charges and allowing the prenyl group to insert into membranes. Another class of geranylgeranylated proteins called Rab proteins does not contain a CAAX box. Geranylgeranylation occurs on one or two cysteines near the COOH terminus. The findings of Glomset and the yeast geneticists fascinated us because of our longstanding interest in the mevalonate pathway, and we were intrigued by its potential connection to cancer (33Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Google Scholar). At this point, we were joined by Yuval Reiss, a postdoctoral fellow from Israel who had worked on ubiquitination with Aaron Ciechanover and Avram Hershko and had mastered the technique of affinity chromatography. Yuval made an affinity column with a CAAX peptide and used it to purify farnesyltransferase from rat liver. The enzyme was a heterodimer composed of an α-subunit that had the transferase activity and a β-subunit that bound to the Ras substrate (37Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides.Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (687) Google Scholar, 38Reiss Y. Seabra M.C. Armstrong S.A. Slaughter C.A. Goldstein J.L. Brown M.S. Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions.J. Biol. Chem. 1991; 266: 10672-10677Abstract Full Text PDF PubMed Google Scholar). Yuval's enzyme was efficient in farnesylating Ras proteins. Most importantly, the enzyme could be inhibited by CAAX peptides as short as four residues (37Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides.Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (687) Google Scholar, 39Goldstein J.L. Brown M.S. Stradley S.J. Reiss Y. Gierasch L.M. Nonfarnesylated tetrapeptide inhibitors of protein farnesyltransferase.J. Biol. Chem. 1991; 266: 15575-15578Abstract Full Text PDF PubMed Google Scholar). Others had shown that farnesylation is essential for the transforming activity of mutant oncogenic Ras proteins. Together with Guy James, a postdoctoral fellow, we immediately began a collaboration with Genentech chemists in an effort to design peptidomimetics that would enter cancer cells and block Ras prenylation (40James G.L. Goldstein J.L. Brown M.S. Rawson T.E. Somers T.C. McDowell R.S. Crowley C.W. Lucas B.K. Levinson A.D. Marsters Jr., J.C. Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells.Science. 1993; 260: 1937-1942Crossref PubMed Google Scholar). Ras proteins come in three varieties. H-Ras is the most heavily studied, but it is not the one that is frequently mutated in human cancers. That distinction belongs to K-Ras, which has a polylysine sequence immediately upstream of the CAAX box. The polylysine sequence gives K-Ras a 50-fold higher affinity for the farnesyltransferase compared with H-Ras (41James G.L. Goldstein J.L. Brown M.S. Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro.J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). James Marsters and his colleagues at Genentech designed a series of benzodiazepine peptidomimetics that were extremely efficient in blocking farnesylation of H-Ras. Unfortunately, these compounds were much less effective in blocking farnesylation of K-Ras. Even more disappointing, we found that when its farnesylation was blocked, K-Ras became an alternative substrate for another enzyme that we characterized, namely geranylgeranyltransferase (now known as GGTase I) (Ref. 41James G.L. Goldstein J.L. Brown M.S. Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro.J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar; see below). Realizing that it would be difficult to prevent all prenylation of K-Ras, we reluctantly gave up our quest to cure cancer. Others persisted, but the clinical results have been disappointing, and currently, there is no approved farnesyltransferase inhibitor in clinical use. Our search for a geranylgeranyltransferase began with the observation by others that geranylgeranyl, not farnesyl, was added to proteins with CAAX boxes terminating in leucine. Chief among these are Rac and Rho proteins that anchor cell membranes to the cytoskeleton. Like Ras proteins, Rho and Rac are GTP-binding proteins. At this point, Yuval Reiss was joined by Miguel Seabra, a graduate student from Portugal. Together, they purified the enzyme that became known as GGTase I. Remarkably, we found that farnesyltransferase and GGTase I share the same catalytic α-subunit. They differ in the β-subunit, which confers the specificity for Ras or Rac/Rho proteins, respectively (42Seabra M.C. Reiss Y. Casey P.J. Brown M.S. Goldstein J.L. Protein farnesyltransferase and geranylgeranyltransferase share a common α-subunit.Cell. 1991; 65: 429-434Abstract Full Text PDF PubMed Scopus (0) Google Scholar). Miguel Seabra went on to make a discovery that was even more intriguing. He set out to purify a third prenyltransferase, namely the enzyme that attaches geranylgeranyl to Rab proteins, a large family of GTP-binding proteins that regulate vesicle fusion reactions. Because Rab proteins do not contain CAAX boxes, Miguel had to use standard column purification techniques. Surprisingly, when he applied a partially purified enzyme preparation to a hydrophobic column, none of the activity was recovered in the eluate. When this happens, the standard approach is to mix together the eluate fractions just in case the enzyme contains two necessary subunits that were separated on the column. This type of mixing experiment almost never works. Nevertheless, we urged Miguel to mix the fractions. All of us were delighted by the result. Indeed, the column had separated two required components, which we called Components A and B (43Seabra M.C. Goldstein J.L. Südhof T.C. Brown M.S. Rab geranylgeranyltransferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys.J. Biol. Chem. 1992; 267: 14497-14503Abstract Full Text PDF PubMed Google Scholar). With this knowledge, Miguel was able to complete the purification of both components, always assaying one component in the presence of the other. Component B contained two polypeptides that roughly resembled in size the α- and β-subunits of farnesyltransferase and GGTase I. Component A was unique (44Seabra M.C. Brown M.S. Slaughter C.A. Südhof T.C. Goldstein J.L. Purification of Component A of Rab geranylgeranyltransferase: possible identity with choroideremia gene product.Cell. 1992; 70: 1049-1057Abstract Full Text PDF PubMed Scopus (0) Google Scholar). Miguel Seabra and Doug Andres, a postdoctoral fellow, then performed a series of kinetic studies that defined the separate roles of Components A and B. It turned out that Component B is indeed the catalytic component (45Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P. Goldstein J.L. cDNA cloning of Component A of Rab geranylgeranyltransferase and demonstration of its role as a Rab escort protein.Cell. 1993; 73: 1091-1099Abstract Full Text PDF PubMed Google Scholar). On its own, Component B attaches a single geranylgeranyl to a single Rab protein. In the absence of Component A, the reaction terminates at that point because the geranylgeranylated Rab remains bound to the enzyme. When Component A is added, it removes the geranylgeranylated Rab from the enzyme and allows the enzyme t
Referência(s)