Botanical dietary supplements are widely used, but issues of authenticity, consistency, safety, and efficacy that complicate their poorly understood mechanism of action have prompted questions and concerns in the popular and scientific literature. Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a multicomponent botanical therapeutic used as a popular remedy for menopause and dysmenorrhea and explored as a treatment in breast and prostate cancer. However, its use and safety are controversial. A. racemosa tissues contain the bioactive serotonin analog N-methylserotonin, which is thought to contribute to the serotonergic activities of black cohosh–containing preparations. A. racemosa has several TDC-like genes hypothesized to encode tryptophan decarboxylases (TDCs) converting L-tryptophan to tryptamine, a direct serotonin precursor in plants. Expression of black cohosh TDC1, TDC2, and TDC3 in Saccharomyces cerevisiae resulted in the production of tryptamine. TDC1 and TDC3 had approximately fourfold higher activity than TDC2, which was attributable to a variable Cys/Ser active site residue identified by site-directed mutagenesis. Co-expression in yeast of the high-activity black cohosh TDCs with the next enzyme in serotonin biosynthesis, tryptamine 5-hydroxylase (T5H), from rice (Oryza sativa) resulted in the production of serotonin, whereas co-expression with low-activity TDCs did not, suggesting that TDC activity is a rate-limiting step in serotonin biosynthesis. Two T5H-like sequences were identified in A. racemose, but their co-expression with the high-activity TDCs in yeast did not result in serotonin production. TDC expression was detected in several black cohosh tissues, and phytochemical analysis using LC-MS revealed several new tryptamines, including tryptamine and serotonin, along with N-methylserotonin and, interestingly, N-N-dimethyl-5-hydroxytryptamine (bufotenine), which may contribute to hepatotoxicity. Incubation of A. racemosa leaves with tryptamine and N-methyltryptamine resulted in increased concentrations of serotonin and N-methylserotonin, respectively, suggesting that methylation of tryptamine precedes hydroxylation in the biosynthesis of N-methylserotonin. This work indicates a significantly greater variety of serotonin derivatives in A. racemosa than previously reported. Moreover, the activities of the TDCs underscore their key role in the production of serotonergic compounds in A. racemosa. Finally, it is proposed that tryptamine is first methylated and then hydroxylated to form the black cohosh signature compound N-methylserotonin.

Many chemicals and cellular processes cause oxidative stress that can damage lipids, proteins, or DNA (1). To quickly sense and respond to this ubiquitous threat, organisms have evolved enzymes that neutralize harmful oxidants such as reactive oxygen species and electrophilic compounds (including xenobiotics and their breakdown products) in cells. These antioxidant enzymes include GSH S-transferase (GST), NADPH:quinone oxidoreductase 1, thioredoxin, hemeoxygenase-1, and others (2, 3). Many of these proteins are commonly expressed in cells exposed to oxidative stress. The antioxidant response element (ARE) is a major regulatory component of this cellular stress response. The ARE is a conserved, 11-nucleotide-long DNA motif present in the 5 -flanking regions of many genes encoding antioxidant proteins. The laboratory ofCecil Pickett (Fig. 1) at theMerck Frosst Centre for Therapeutic Research in Quebec discovered ARE, a finding reported in the early 1990s in two JBC papers recognized as Classics here (4, 5). ARE’s discovery was spurred in large part by Pickett’s career choice. After completing a PhD in biology and a 2-year postdoc at UCLA in themid-1970s, he began towork in the pharmaceutical industry. Recruited to Merck in 1978 by its then head of research and development (and later CEO), Roy Vagelos, “I became interested in how drug-metabolizing enzymes were induced by various xenobiotics,” Pickett says. According to Pickett, Vagelos encouraged researchers at the company “to really start a program where we could build our research careers. But he also said, ‘Keep in mind the long-term mission ofMerck—and that is to discover novel medicines that could help people.’” Pickett remembers joining Merck at a unique time in the pharmaceutical industry. “[Vagelos] really had an incredible vision for research and development.” Inspired by Vagelos’ vision, Pickett launched a vigorous research program that attracted many talented scientists. To find xenobiotic-metabolizing enzymes, Pickett and his team treated rats with various chemicals, including phenobarbital and 3-methylcholanthrene, to induce genes involved in xenobiotic metabolism and breakdown. To isolate these genes, the team used an in vitro system for translation of the mRNAs isolated from the livers of the animals. This approach yielded promising results. “I noticed that the profile of the in vitro translated material from induced animals was very different from that of noninduced animals,” says Pickett. “And there tended to be a low-molecular-weight protein that was always induced.” At first, it was unclear what this small proteinmight be. But a colleague at Merck, Anthony Lu, had a hunch that it might be GST, Pickett says. Acting on Lu’s idea, Pickett and his team applied a technique called polysomal immunoprecipitation. The researchers used GST-specific antibodies from Barbara Hales’ laboratory at McGill University in Montreal to detect and isolate mRNAribosome complexes that contained GST-encoding mRNA sequences (6). “That allowed us to synthesize cDNAs and isolate the structural genes for the GSTs,” says Pickett. JBC Deputy Editor F. Peter Guengerich at Vanderbilt University nominated this paper as a Classic. 1 Martin Spiering is the technical editor at JBC. E-mail: mspiering@asbmb.org. 2 The abbreviations used are: GST, GSH S-transferase; ARE, antioxidant response element; Ah, aryl hydrocarbon; XRE, xenobiotic-responsive element; NRF2, NFE2-related factor 2. Figure 1. Cecil Pickett (pictured) and colleagues first described the ARE motif, present in the 5 regions of many genes whose expression is upregulated by oxidative stress and xenobiotics. Photo courtesy of Cecil Pickett. CLASSICS

Molecular oxygen is essential for the viability and function of every cell of the animal body. Because skin, tissues, and organs impede oxygen diffusion directly from the air, molecular oxygen concentrations inside the body often are less than 5% (1Semenza G.L. Agani F. Booth G. Forsythe J. Iyer N. Jiang B.H. Leung S. Roe R. Wiener C. Yu A. Structural and functional analysis of hypoxia-inducible factor 1.Kidney Int. 1997; 51 (OnlinePDF 9027737): 553-555Abstract Full Text PDF PubMed Scopus (232) Google Scholar), much lower than the 21% in Earth's atmosphere. As multicellular organisms evolved, this steep oxygen gradient necessitated the development of structures such as the circulatory system and of biochemical mechanisms that monitor and control oxygen levels in the body. A major player in sensing potentially harmful drops in cellular oxygen concentrations (hypoxia) is the transcription factor hypoxia-inducible factor 1 (HIF-1). 2The abbreviations used are: HIFhypoxia-inducible factorPHDprolyl-4-hydroxylase domain. 2The abbreviations used are: HIFhypoxia-inducible factorPHDprolyl-4-hydroxylase domain. HIF-1, and its close relative HIF-2, regulate many genes, including the erythropoietin (EPO) gene, which encodes a hormone that stimulates production of red blood cells (2Semenza G.L. Nejfelt M.K. Chi S.M. Antonarakis S.E. Hypoxia-inducible nuclear factors bind to an enhancer element located 3′ to the human erythropoietin gene.Proc. Natl. Acad. Sci. U.S.A. 1991; 88 (OnlinePDF 2062846): 5680-5684Crossref PubMed Scopus (648) Google Scholar, 3Semenza G.L. Wang G.L. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.Mol. Cell. Biol. 1992; 12 (OnlinePDF 1448077): 5447-5454Crossref PubMed Scopus (2015) Google Scholar). hypoxia-inducible factor prolyl-4-hydroxylase domain. hypoxia-inducible factor prolyl-4-hydroxylase domain. One important milestone in uncovering HIF-1's pivotal role in oxygen sensing was its purification and biochemical characterization in the mid-1990s by the lab of Gregg Semenza (Fig. 1), a geneticist at Johns Hopkins University School of Medicine. This work was reported in two JBC papers recognized as Classics here (4Wang G.L. Semenza G.L. Purification and characterization of hypoxia-inducible factor 1.J. Biol. Chem. 1995; 270 (OnlinePDF 7836384): 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1587) Google Scholar, 5Jiang B.H. Rue E. Wang G.L. Roe R. Semenza G.L. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.J. Biol. Chem. 1996; 271 (OnlinePDF 8663540): 17771-17778Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). “JBC was the first choice,” says Semenza, referring to the publication of the first Classics article. “To me, that was a classic JBC paper.” A few years before this work, Semenza's team had found that hypoxia induces the binding of a nuclear protein to a 50-nucleotide-long enhancer region located in the 3′-flanking region of the EPO gene (2Semenza G.L. Nejfelt M.K. Chi S.M. Antonarakis S.E. Hypoxia-inducible nuclear factors bind to an enhancer element located 3′ to the human erythropoietin gene.Proc. Natl. Acad. Sci. U.S.A. 1991; 88 (OnlinePDF 2062846): 5680-5684Crossref PubMed Scopus (648) Google Scholar, 3Semenza G.L. Wang G.L. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.Mol. Cell. Biol. 1992; 12 (OnlinePDF 1448077): 5447-5454Crossref PubMed Scopus (2015) Google Scholar). Although the researchers could delineate the DNA region that is bound by the hypoxia-induced protein, the protein's identity was unknown, prompting Semenza to look for it. Semenza's team first had to cross some unexpectedly rough waters. “We used an expression cloning strategy,” he says. The researchers expressed human proteins from cDNAs in bacteriophages and then used the HIF-binding oligonucleotide as a probe to find proteins binding to it. “We screened millions and millions of clones and got nothing.” What went wrong? “We had initially performed experiments that suggested that there was a single subunit that could bind to the DNA fragment,” says Semenza. As the later results of the two Classics papers revealed, HIF-1 is a heterodimeric protein (4Wang G.L. Semenza G.L. Purification and characterization of hypoxia-inducible factor 1.J. Biol. Chem. 1995; 270 (OnlinePDF 7836384): 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1587) Google Scholar, 5Jiang B.H. Rue E. Wang G.L. Roe R. Semenza G.L. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.J. Biol. Chem. 1996; 271 (OnlinePDF 8663540): 17771-17778Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar), a fact that probably hobbled the bacteriophage-based approach, since each phage clone typically contains only a single gene. Semenza and his team were undeterred. “We could give up and let someone else do it—but that did not sound like a good idea,” says Semenza. The team changed tack, embarking on a biochemical purification. They grew HeLa cells in large-scale (>100-liter) cultures, exposed them to hypoxia and cobalt chloride (which also induces HIF activity), prepared nuclear extracts from them, and purified HIF-1 via DNA-affinity chromatography with an oligonucleotide that contained the HIF-binding site. After isolating the HIF-1 proteins in a preparative gel-shift assay along with glycerol sedimentation, the authors could show that HIF-1 is composed of two subunits: a larger one of 120 kDa, called HIF-1α, and a smaller one of 91–94 kDa, called HIF-1β (4Wang G.L. Semenza G.L. Purification and characterization of hypoxia-inducible factor 1.J. Biol. Chem. 1995; 270 (OnlinePDF 7836384): 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1587) Google Scholar). The purification yielded enough protein of the two HIF-1 subunits for amino acid microsequencing that gave short protein sequences. The researchers used that sequence information in a cloning approach to identify and sequence full-length cDNAs of the HIF-1 subunits (6Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc. Natl. Acad. Sci. U.S.A. 1995; 92 (OnlinePDF 7539918): 5510-5514Crossref PubMed Scopus (4674) Google Scholar). The cDNA sequences showed that both HIF-1 subunits belong to a group of transcription factors containing a basic helix-loop-helix (bHLH) motif required for dimerization and DNA binding and a Per-Arnt-Sim (PAS) domain for protein–protein interactions. The HIF-1β sequence was identical to that of a previously identified protein, aryl hydrocarbon receptor nuclear translocator (ARNT), a subunit of the dioxin receptor, which responds to dioxins and other toxic chemicals. However, the HIF-1α sequence was previously unknown and only partially similar to single-minded homolog (Sim), a transcription factor in Drosophila. The second Classics paper (5Jiang B.H. Rue E. Wang G.L. Roe R. Semenza G.L. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.J. Biol. Chem. 1996; 271 (OnlinePDF 8663540): 17771-17778Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar) helped define the regions in HIF-1α and HIF-1β required for dimerization of the two subunits, DNA binding, and activation of transcription of hypoxia-induced genes. What might now look like a series of straightforward steps of purifying and sequencing a protein was more an exercise in continual improvisation. “My lab was a molecular genetics laboratory,” says Semenza. “We did not even own a fraction collector,” required for protein purification. Luckily for Semenza, the lab of another Johns Hopkins researcher, Thomas Kelly, was just across the street. “Tom was one of the first people to purify a protein based on its binding to DNA,” notes Semenza, giving his project a vital boost. “We could not have [isolated HIF-1] without the help from Tom Kelly's laboratory.” The biochemical characterization of HIF-1 by Semenza and colleagues threw open the doors to many additional studies from Semenza's group and also other labs. What emerged from these investigations is that the specific HIF-1–binding site is ubiquitous in the human genome. It's been found at many locations, including the 5′-flanking regions and introns of many genes, says Semenza. This hinted at HIF-1's role as the central hub of metabolic regulation in response to oxygen. “These genes are not induced by hypoxia in every cell type under every condition,” Semenza notes. “They're induced by hypoxia in some cell types under some conditions—the plasticity of the [hypoxic] response is remarkable.” The HIF-1–regulated genes encode proteins in many metabolic processes, including glycolysis, angiogenesis, and wound healing. They are also expressed when people move from lower to higher altitudes, and they are hyperactive in some diseases, most notably cancer (7Semenza G.L. Hypoxia-inducible factors in physiology and medicine.Cell. 2012; 148 (OnlinePDF 22304911): 399-408Abstract Full Text Full Text PDF PubMed Scopus (1669) Google Scholar, 8Nakazawa M.S. Keith B. Simon M.C. Oxygen availability and metabolic adaptations.Nat. Rev. Cancer. 2016; 16 (OnlinePDF 27658636): 663-673Crossref PubMed Scopus (171) Google Scholar). Consequently, HIF-mediated signaling is now a therapeutic target to combat cancer and manage disorders such as altitude sickness. A key feature of HIF-1 activity is its regulation by specific prolyl-4-hydroxylase domain (PHD) proteins. In the presence of normal oxygen concentration, the PHDs hydroxylate one or both of two conserved proline residues in HIF-1α, leading to binding by the von Hippel-Lindau protein (VHL), which targets HIF-1α for proteasomal degradation (9Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway.Mol. Cell. 2008; 30 (OnlinePDF 18498744): 393-402Abstract Full Text Full Text PDF PubMed Scopus (1908) Google Scholar). “So [cells] make HIF1α, but it's being degraded when oxygen is available,” explains Semenza. When oxygen levels become limiting, the PHD proteins are inhibited, increasing the fraction of HIF-1α that's not hydroxylated. This stabilizes the HIF-1 protein, causing its rapid accumulation and leading to the activation of its target genes. “It is a really beautiful system,” says Semenza. For his work on the role of HIF-1 in oxygen sensing, Semenza was awarded the Nobel Prize in Physiology or Medicine in 2019 (shared with William G. Kaelin, Jr., and Peter J. Ratcliffe for their work on related oxygen-sensing pathways) and the Albert Lasker Award for Basic Medical Research in 2016.

Macromolecules such as proteins, lipids, and carbohydrates often have complex structures that underpin their cellular functions. The sugar alcohol myo-inositol is a notable exception—its simple six-carbon structure is rather unremarkable (Fig. 1) but is used in countless cellular processes in all domains of life (1Michell R.H. Inositol lipids: from an archaeal origin to phosphatidylinositol 3,5-bisphosphate faults in human disease.FEBS J. 2013; 280 (23902363): 6281-629410.1111/febs.12452Crossref PubMed Scopus (36) Google Scholar). For instance, inositol provides the structural backbone of phosphatidylinositol, an essential building block of important signaling molecules such as inositol polyphosphates. Phosphatidylinositol is one of the most abundant membrane phospholipids in eukaryotes, vital for glycosylphosphatidylinositol-anchored proteins, and a component of some sphingolipids (2Henry S.A. Kohlwein S.D. Carman G.M. Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae.Genetics. 2012; 190 (22345606): 317-34910.1534/genetics.111.130286Crossref PubMed Scopus (302) Google Scholar, 3Henry S.A. Gaspar M.L. Jesch S.A. The response to inositol: regulation of glycerolipid metabolism and stress response signaling in yeast.Chem. Phys. Lipids. 2014; 180 (24418527): 23-4310.1016/j.chemphyslip.2013.12.013Crossref PubMed Scopus (53) Google Scholar). “The fact of the matter is that inositol is absolutely essential [in cells],” says Susan Henry, Professor of Molecular Biology and Genetics at Cornell University (Fig. 2). Henry has studied inositol metabolism in the yeast Saccharomyces cerevisiae since the 1970s. “I focused on the phospholipids and the metabolites that regulate their formation,” she says. “Inositol turned out to be the strongest regulatory metabolite of these pathways.” S. cerevisiae was Henry's organism of choice almost immediately from the start of her long career, she says, because it is easier to study inositol and phospholipids in this species than in more complex eukaryotes. Yeast species have been a workhorse for scientists since the dawn of modern research. Their widespread use in fermentations led to the coinage of the term enzyme (Greek for in yeast) (4Robinson P.K. Enzymes: principles and biotechnological applications.Essays Biochem. 2015; 59 (26504249): 1-4110.1042/bse0590001Crossref PubMed Google Scholar). S. cerevisiae grows rapidly in culture and does so as single cells, a boon for investigating eukaryotic biochemistry under controlled conditions. The species can be stably maintained in the haploid state, and its genes can be easily manipulated. “Yeast is almost like the Escherichia coli of the eukaryotic world. [It helped us] to figure out exactly where the metabolic components are coming from,” says Henry. The fully sequenced S. cerevisiae genome did not become available until 1996 (5Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnston M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin H. Oliver S.G. Life with 6000 genes.Science. 1996; 274 (8849441): 546-56710.1126/science.274.5287.546Crossref PubMed Scopus (3052) Google Scholar), so earlier studies of the genetics and biochemistry even of simple organisms such as yeast required skilled detective work to find all the players involved in a molecular pathway. While at Albert Einstein College of Medicine in the mid-1970s, Henry and Ph.D. student Michael Culbertson used the mutagenic agent ethyl methanesulfonate to generate a series of more than 50 S. cerevisiae mutants defective in inositol biosynthesis (6Culbertson M.R. Henry S.A. Inositol-requiring mutants of Saccharomyces cerevisiae.Genetics. 1975; 80 (1093935): 23-40Crossref PubMed Google Scholar). This mutant strain collection provided a resource to launch investigations into the genes involved in inositol and phospholipid metabolism. In a 1981 JBC paper, Henry and co-author Thomas Donahue reported the first purification and characterization of yeast myo-inositol-1-phosphate synthase (7Donahue T.F. Henry S.A. myo-Inositol-1-phosphate synthase: characteristics of the enzyme and identification of its structural gene in yeast.J. Biol. Chem. 1981; 256 (7016881): 7077-7085Abstract Full Text PDF PubMed Google Scholar). This enzyme, called Ino1, is an intramolecular lyase and isomerase that catalyzes the cyclization of glucose 6-phosphate, a reaction that yields inositol 1-phosphate, an immediate precursor to free inositol. The two scientists also mapped its gene to a locus (called INO1) in the yeast genome and developed antibodies for specific detection of Ino1, laying the groundwork for more detailed biochemical and genetic studies. In three JBC papers published in the late 1980s and early 1990s and recognized as Classics here (8Dean-Johnson M. Henry S.A. Biosynthesis of inositol in yeast: primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the ino1 locus.J. Biol. Chem. 1989; 264 (2642902): 1274-1283Abstract Full Text PDF PubMed Google Scholar, 9White M.J. Hirsch J.P. Henry S.A. The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper.J. Biol. Chem. 1991; 266 (1985968): 863-872Abstract Full Text PDF PubMed Google Scholar, 10Ambroziak J. Henry S.A. INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter.J. Biol. Chem. 1994; 269 (8195172): 15344-15349Abstract Full Text PDF PubMed Google Scholar), Henry and colleagues at Albert Einstein College and at Carnegie Mellon University reported the sequence and genetic analysis of the INO1 gene, along with its regulation by a transcriptional repressor and two transcriptional activators. In the first paper (8Dean-Johnson M. Henry S.A. Biosynthesis of inositol in yeast: primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the ino1 locus.J. Biol. Chem. 1989; 264 (2642902): 1274-1283Abstract Full Text PDF PubMed Google Scholar), Henry and Margaret Dean-Johnson sequenced the cloned INO1 gene, including its 5′ and 3′ regulatory regions, and also determined the amino acid sequence of the purified protein. This analysis uncovered an ORF as a prime candidate for encoding the entire enzyme. When they disrupted the predicted INO1 ORF in yeast cells, the researchers found that the cells did not express any protein detectable by the antibodies for Ino1 and that the cells grew only when supplied with inositol from the growth medium. The findings showed that the INO1 gene encodes myo-inositol-1-phosphate synthase in yeast, representing a major advance because it made available the full-length nucleotide and amino acid sequences of this central phospholipid enzyme in eukaryotes (11Majumder A.L. Johnson M.D. Henry S.A. 1l-myo-inositol-1-phosphate synthase.Biochim. Biophys. Acta. 1997; 1348 (9370339): 245-25610.1016/s0005-2760(97)00122-7Crossref PubMed Scopus (162) Google Scholar). In keeping with earlier findings of Henry's laboratory that expression of Ino1 is transcriptionally regulated (12Hirsch J.P. Henry S.A. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.Mol. Cell. Biol. 1986; 6 (3025587): 3320-332810.1128/MCB.6.10.3320Crossref PubMed Scopus (179) Google Scholar), the Classics paper also uncovered several conserved short DNA motifs in the 5′ promoter region of the INO1 gene that were likely binding sites of transcriptional regulators (8Dean-Johnson M. Henry S.A. Biosynthesis of inositol in yeast: primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the ino1 locus.J. Biol. Chem. 1989; 264 (2642902): 1274-1283Abstract Full Text PDF PubMed Google Scholar). Henry therefore next set her sights on deciphering the regulation of INO1 expression by inositol and another phospholipid precursor, choline. Using different INO1 promoter constructs fused to the E. coli lacZ gene to measure the promoters' activities, her team pinpointed the main transcriptional start site, a TATA box, and several transcription factor–binding sites in the INO1 promoter (13Lopes J.M. Hirsch J.P. Chorgo P.A. Schulze K.L. Henry S.A. Analysis of sequences in the INO1 promoter that are involved in its regulation by phospholipid precursors.Nucleic Acids Res. 1991; 19 (2027776): 1687-169310.1093/nar/19.7.1687Crossref PubMed Scopus (99) Google Scholar). The team also found a region that appeared to be bound by a transcriptional repressor, Opi1 (named after the overproduction of inositol phenotype of yeast strains lacking this repressor), which they had previously identified (14Bailis A.M. Lopes J.M. Kohlwein S.D. Henry S.A. Cis and trans regulatory elements required for regulation of the CHO1 gene of Saccharomyces cerevisiae.Nucleic Acids Res. 1992; 20 (1313970): 1411-141810.1093/nar/20.6.1411Crossref PubMed Scopus (53) Google Scholar). In the second Classics paper (9White M.J. Hirsch J.P. Henry S.A. The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper.J. Biol. Chem. 1991; 266 (1985968): 863-872Abstract Full Text PDF PubMed Google Scholar), Henry and colleagues mapped the OPI1 gene in the yeast genome, cloned and sequenced it, and identified key features of the predicted Opi1 protein sequence, including a leucine repeat and polyglutamine stretches also present in other regulatory proteins. The paper defined a major regulatory mechanism that controls INO1 expression. It also provided critical momentum for work by Henry's group that later identified a cis-acting regulatory DNA element, the inositol-sensitive upstream activation sequence (UASINO). This sequence is present in the promoters of genes responsible for the synthesis of phospholipids and also the lipid triacylglycerol (14Bailis A.M. Lopes J.M. Kohlwein S.D. Henry S.A. Cis and trans regulatory elements required for regulation of the CHO1 gene of Saccharomyces cerevisiae.Nucleic Acids Res. 1992; 20 (1313970): 1411-141810.1093/nar/20.6.1411Crossref PubMed Scopus (53) Google Scholar). The third Classics paper capped off the series by further elucidating the regulatory circuit that controls phospholipid biosynthesis in yeast (10Ambroziak J. Henry S.A. INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter.J. Biol. Chem. 1994; 269 (8195172): 15344-15349Abstract Full Text PDF PubMed Google Scholar). Henry and colleagues demonstrated that the yeast proteins Ino2 and Ino4 form a heterodimeric complex that binds and activates the INO1 promoter. They also delineated the binding sites of the Ino2-Ino4 complex on this promoter and showed that both proteins contain a basic helix-loop-helix motif characteristic of transcriptional regulators. The paper was the first to describe a basic helix-loop-helix heterodimeric transcription factor in yeast and represented a milestone in the then budding field of research into the regulation of phospholipid synthesis in eukaryotes. Looking back, Henry says that the mentorship by two of her early advisors, Seymour Fogel and Alec Keith, helped lay the foundation for her career. Besides sharing their expertise in genetics and biochemistry, they also gave Henry critical material support to get her work off the ground. “I was really lucky that they were not the kind of people who wanted me to [work exclusively] on their hot project,” she says. “They were willing to let me come into the laboratory and use their materials to do the things that I wanted to do.” Moreover, although Henry was working with yeast, she could secure funding through agencies that typically support mainly medical research, she says. “I did not have any trouble getting support from the National Institutes of Health, because of the connection with lipid metabolism in other eukaryotic organisms.” This investment paid off well, she notes. “Many of the genes that I worked on were homologous to those in other eukaryotes, providing a ladder for other people to find the [corresponding] genes in other organisms.”

Lipid droplets (LDs) are key lipid storage structures in cells. One might think that they simply result from lipid molecules separating out within the aqueous intracellular environment. However, closer investigation has revealed that LDs are wellorganized organelles surrounded by proteins that tightly control lipid entry into and exit from these organelles. One LD-associated protein is perilipin 1 (PLIN1) (1). It is a highly abundant protein in fat cells (adipocytes) and the first protein identified on the LD surface (Fig. 1), as reported in a JBC paper recognized as a Classic here (2), authored by Andrew Greenberg (Fig. 2), his late mentor Constantine Londos, and colleagues at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. JBC reached out to Greenberg to learn about the discovery of PLIN1 and how this research milestone has spurred further investigations into fat storage andmetabolism.

The sexes in humans and other animals typically display numerous differences. This belies the fact that the two major hormone classes responsible for these differences, androgens and estrogens, differ only subtly in their chemical backbones. Androgens have a six-carbon nonaromatic ring—the “A ring” (Fig. 1)—in their steroid skeleton, whereas estrogens have an aromatic A ring. Remarkably, a single protein, steroid aromatase (also called estrogen synthase), is the only known enzyme capable of “aromatizing” the A ring in androgens such as testosterone and androstenedione to produce estrogens such as estradiol and estrone. Females and males require both sex hormones in a balance appropriate for each sex. A chief role of steroid aromatase, along with those of other enzymes that modify or metabolize steroid hormones, is to maintain this healthy balance, for example, during development and pregnancy and in reproductive tissues. Accordingly, researchers have long sought to better understand steroid aromatase activity in order to reduce pregnancy complications, develop hormone-based contraceptives, and manage estrogen-responsive cancers. Three papers published in the Journal of Biological Chemistry, authored by Kenneth J. Ryan and recognized as Classics here (1Ryan K.J. Engel L.L. Hydroxylation of steroids at carbon 21.J. Biol. Chem. 1957; 225 (13416221): 103-114http://www.jbc.org/content/225/1/103.longAbstract Full Text PDF PubMed Google Scholar, 2Ryan K.J. Biological aromatization of steroids.J. Biol. Chem. 1959; 234 (13630892): 268-272http://www.jbc.org/content/234/2/268.longAbstract Full Text PDF PubMed Google Scholar3Ryan K.J. Metabolism of C-16–oxygenated steroids by human placenta: the formation of estriol.J. Biol. Chem. 1959; 234 (13673004): 2006-2008http://www.jbc.org/content/234/8/2006.longAbstract Full Text PDF PubMed Google Scholar), laid the groundwork for understanding the role of steroid aromatase and other steroid-modifying enzymes in estrogen biosynthesis. Before embarking on his studies of steroid aromatase, Ryan, along with co-author Lewis Engel, discovered that microsomal fractions of adrenal glands from beef contain an enzyme activity that hydroxylates carbon 21 in the hormone progesterone and several of its derivatives (1Ryan K.J. Engel L.L. Hydroxylation of steroids at carbon 21.J. Biol. Chem. 1957; 225 (13416221): 103-114http://www.jbc.org/content/225/1/103.longAbstract Full Text PDF PubMed Google Scholar). This finding clarified the enzymatic nature of this pivotal step in steroid hormone production in adrenal tissues. It revealed that this step consists of an oxidative reaction that requires microsomes, NADPH, and molecular oxygen. It also helped establish an experimental system that Ryan could then use to probe how estrogens are made from androgens. “[He was] trying to figure out the physiology of pregnancy, what the estrogens were doing, and how they how they arose,” says Richard Auchus, Professor of Internal Medicine and Pharmacology at the University of Michigan and a researcher with a long-standing research interest in steroid production and modification. The in vivo conversion of an androgen to estrogenic activity was noted more than 80 years ago, when Steinach and Kun (4Steinach E. Kun H. Transformation of male sex hormones into a substance with the action of a female hormone.Lancet. 1937; 133: 845Abstract Scopus (22) Google Scholar), using a bioassay that measured hormone activities only indirectly, reported that men given testosterone have higher estrogenic activity in urine. This finding suggested that estrogens can be formed from androgens, and further studies in the 1950s with extracts from animal tissues hinted at the enzymatic nature of this conversion (5Meyer A.S. Conversion of 19-hydroxy-Δ4-androstene-3,17-dione to estrone by endocrine tissue.Biochim. Biophys. Acta. 1955; 17 (13239703): 441-44210.1016/0006-3002(55)90395-4Crossref PubMed Scopus (117) Google Scholar, 6Meyer A.S. Hayano M. Lindberg M.C. Gut M. Rodgers O.G. The conversion of Δ4-androstene-3,17-dione-4-C14 and dehydroepiandrosterone by bovine adrenal homogenate preparations.Acta Endocrinol. 1955; 18 (14349559): 148-16810.1530/acta.0.0180148Crossref PubMed Google Scholar). However, the activities in these preparations were insufficient to study the nature of the androgen-to-estrogen conversion and its roles in human health and reproduction. This dearth in available methods to investigate this conversion prompted Ryan, then a clinician-researcher at Harvard Medical School, to try to boost estrogen production, extract these hormones more efficiently, and begin to characterize the enzymes involved. “He was one of the first people who did that sort of stuff,” says Auchus. Moreover, Ryan was an obstetrician, meaning that he had a ready supply of tissue ideal for investigating estrogen production—human placentas obtained immediately after delivery. Preparing and analyzing these biological materials was laborious, requiring many time-consuming steps now seldom performed in biochemistry labs. Ryan had to carefully extract the enzyme activities from kilograms of placental tissues, and even small glitches in the experimental protocols could result in loss of activity after hours of preparation. “These were some of the first very brave steps to try to figure out where [estrogens] were coming from,” says Auchus. This effort bore fruit when, in 1959, Ryan confirmed that the formation of the aromatic A ring is the result of enzymatic activity leading to aromatization (2Ryan K.J. Biological aromatization of steroids.J. Biol. Chem. 1959; 234 (13630892): 268-272http://www.jbc.org/content/234/2/268.longAbstract Full Text PDF PubMed Google Scholar). “He figured out that [the enzyme activity] was in the microsomal fraction and that it required NADPH, and he developed the chromatography to measure it,” Auchus says. Ryan's findings suggested that the aromatization involved an enzyme system that used molecular oxygen to achieve A-ring aromatization—yet, quite paradoxically, no extra oxygen atoms were retained in the estrogen products. With the tools available at the time, Ryan could not delineate the exact biochemical sequence of events, nor could he isolate the enzyme(s) responsible or the reaction intermediates that bore the tell-tale oxygen atoms. According to Auchus, initially it wasn't even clear whether the aromatization was performed by one enzyme or by several because the reaction sequence required at least three oxidations, which yielded some, at the time, unusual products. “[It] was fascinating that you got an aromatic A ring, [but] this did not really make a lot of sense to people back then.” says Auchus. “The reaction was weird because it involved both loss of a methyl group and formation of the aromatic A ring.” The reactions remained black boxes, but Ryan's work had begun to pry the lids open. “Now people could start to look at the mechanism,” says Auchus. Studies in the 1980s revealed that the aromatizing activity is indeed performed by just one enzyme, a cytochrome P450 monooxygenase (7Mendelson C.R. Wright E.E. Evans C.T. Porter J.C. Simpson E.R. Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification.Arch. Biochem. Biophys. 1985; 243 (4083898): 480-49110.1016/0003-9861(85)90525-9Crossref PubMed Scopus (145) Google Scholar, 8Kellis J.T. Vickery L.E. Purification and characterization of human placental aromatase cytochrome P-450.J. Biol. Chem. 1987; 262 (3104339): 4413-4420http://www.jbc.org/content/262/9/4413.longAbstract Full Text PDF PubMed Google Scholar9Osawa Y. Yoshida N. Fronckowiak M. Kitawaki J. Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1β and 2β-monohydroxylation, and recognition of aromatase isozymes.Steroids. 1987; 50 (3142109): 11-2810.1016/0039-128X(83)90058-2Crossref PubMed Scopus (71) Google Scholar). This enzyme, aptly named steroid aromatase, oxidizes carbon 19 (a methyl group attached to the A ring in androgens) in a multistep reaction sequence, starting with two sequential hydroxylations and culminating in an unusual C–C bond cleavage, leading to C-19 elimination and formation of the phenolic A ring in estrogens (Fig. 1). However, as Auchus notes, the details of the third aromatase-catalyzed step remained long unresolved and were somewhat controversial. “People fairly quickly figured out where the first two reactions occurred, but the third reaction [resulting in formic acid release from carbon 19 and the A-ring aromatization] was a vexing step that people could not work out for a long time.” Recent work has now shown that this reaction involves abstraction of a hydrogen atom from carbon 1 of the 19-gem-diol, rearrangement with loss of the formic acid prior to oxygen rebound, and, finally, keto-enol tautomerization to form the phenolic A ring (10Yoshimoto F.K. Guengerich F.P. Mechanism of the third oxidative step in the conversion of androgens to estrogens by cytochrome P450 19A1 steroid aromatase.J. Am. Chem. Soc. 2014; 136 (25252141): 15016-1502510.1021/ja508185dCrossref PubMed Scopus (79) Google Scholar). “Suffice it to say, it is a unique chemical reaction,” Auchus concludes. Ryan next set his sights on establishing the biochemical origins of another important estrogen, estriol, now a standard biomarker in routine pregnancy care. One classic pathway of estriol formation involved the hydroxylation of carbon 16 in the estrogens estradiol and estrone. However, in an earlier study, Ryan had found preliminary evidence for another pathway in which estriol also could be produced by aromatization of C-16–hydroxylated androgens (11Ryan K.J. Conversion of Δ5-androstene-3β,16α,17β-triol to estriol by human placenta.Endocrinology. 1958; 63 (13574097): 392-39410.1210/endo-63-3-392Crossref PubMed Scopus (8) Google Scholar). Using his placental microsomal enzyme system, Ryan confirmed his earlier finding of estriol production from 16α-testosterone. He also clarified that another estrogen, 16α-hydroxyestrone, is an intermediate in the classic estriol-producing pathway (3Ryan K.J. Metabolism of C-16–oxygenated steroids by human placenta: the formation of estriol.J. Biol. Chem. 1959; 234 (13673004): 2006-2008http://www.jbc.org/content/234/8/2006.longAbstract Full Text PDF PubMed Google Scholar). These discoveries underscored the utility of Ryan's assay and represented key early steps in untangling the biochemical complexities in estrogen production. Born in 1926 in New York City, Ryan grew up during the Great Depression, working on farms in his teen years and, following graduation from high school, serving in the United States Navy during World War II (12Schiff I. Frigoletto F. Obituary for Kenneth J. Ryan, M.D.Fertil. Steril. 2002; 78: 1136-113710.1016/S0015-0282(02)04235-8Abstract Full Text Full Text PDF Google Scholar). Upon returning from the war, he enrolled in Northwestern University for his undergraduate studies and then joined Harvard Medical School, graduating magna cum laude in 1952. During a couple of residencies at hospitals in the Boston area, Ryan landed a biochemistry fellowship shared with Nobel Laureate Fritz Lipmann, enabling him to pursue his interest in the roles of estrogens in the biology of pregnancy. In the 1960s, thanks to his managerial skills and knack for anticipating future directions in clinical research, Ryan began taking on administrative duties, successively becoming chairman of several obstetrics and gynecology departments across the country. In the early 1970s, he returned to Harvard where he helped build an academic OB-GYN department. In addition to his work in the clinic and extensive research activities, Ryan trained and mentored many students and residents. He also became active in medical ethics. “He actually got involved with a lot of ethical issues, like fetal tissue research,” Auchus says. “In his later years, he became a pretty prominent person in that field.” Notably, Ryan chaired the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. This commission produced the Belmont Report in 1978, whose guidelines for protecting the rights and dignity of human subjects in research continue to provide an ethical framework for research and health providers in the United States to this day. Ryan also was an early and strong proponent of reproductive choice. He died in 2002 at the age of 75.

Healthy cells are expert recyclers, rapidly breaking down worn-out or surplus macromolecules and reusing their building blocks. Several pathways, such as the proteasomal degradation route for protein breakdown, specifically pick out damaged or expendable molecules. These selective degradation pathways usually operate in cells that have ready access to nutrients. But when cells encounter severe crises such as nutrient shortages, they activate an emergency recycling mechanism. In these cells, a bulk degradation pathway indiscriminately breaks down macromolecules and entire cytoplasmic organelles and ribosomes. This process is commonly referred to as autophagy (Greek for “self-eating”) or sometimes as macroautophagy because other more specialized forms of autophagy are more restricted and selective (1Thorburn A. Autophagy and disease.J. Biol. Chem. 2018; 293 (OnlinePDF 29191833): 5425-5430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 2Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147 (OnlinePDF 22078875): 728-741Abstract Full Text Full Text PDF PubMed Scopus (3917) Google Scholar). It can be thought of as the rough equivalent of a wood chipper that shreds chairs, tables, and credenzas wholesale for use in plywood or as fireplace fuel. Autophagy also represents an important quality-control mechanism that eliminates long-lived proteins and damaged organelles in some cells, such as neurons (2Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147 (OnlinePDF 22078875): 728-741Abstract Full Text Full Text PDF PubMed Scopus (3917) Google Scholar), whose total destruction by processes such as apoptosis would harm the organism. Defects in autophagy have been linked to human disorders, such as neurodegenerative diseases and cancer (1Thorburn A. Autophagy and disease.J. Biol. Chem. 2018; 293 (OnlinePDF 29191833): 5425-5430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 2Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147 (OnlinePDF 22078875): 728-741Abstract Full Text Full Text PDF PubMed Scopus (3917) Google Scholar), highlighting that this recycling mechanism is essential for keeping cells healthy and alive. However, how a cell's nutrient status is communicated to the autophagy machinery was unknown for quite some time. In a 1998 landmark paper published in the Journal of Biological Chemistry and now recognized as a Classic here, Takeshi Noda and Yoshinori Ohsumi (Fig. 1) at the National Institute for Basic Biology in Japan uncovered this missing link, reporting that the target of rapamycin (Tor) 2The abbreviations used are: Tortarget of rapamycinALPalkaline phosphatase. protein in yeast suppresses autophagy in cells that are growing in nutrient-rich conditions (3Noda T. Ohsumi Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast.J. Biol. Chem. 1998; 273 (OnlinePDF 9461583): 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). target of rapamycin alkaline phosphatase. Researchers had known since the early 1990s that two Tor proteins of budding yeast (Saccharomyces cerevisiae), Tor1 and Tor2, respond to the nutritional state of the cell and regulate cell cycle progression (4Lorberg A. Hall M.N. TOR: the first 10 years.Curr. Top. Microbiol. Immunol. 2004; 279 (OnlinePDF 14560948): 1-18Crossref PubMed Google Scholar). Noda and Ohsumi's discovery that Tor proteins also control autophagy represented a major milestone in unraveling how bulk degradation is regulated in eukaryotic cells. Ohsumi had begun studying autophagy in yeast also in the early 1990s (5Ohsumi, Y., (2016) Molecular mechanisms of autophagy in yeast. Nobel Lecture, December 7, 2016 OnlinePDF.Google Scholar). He chose this organism in part because it has a large vacuole (a structure analogous to the lysosome in mammalian cells) that is easy to study by light microscopy. In 1992, his team discovered that when yeast mutant cells that cannot degrade proteins via the proteasomal pathway are starved of nutrients, their vacuoles quickly fill up with conspicuous spherical structures (6Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.J. Cell Biol. 1992; 119 (OnlinePDF 1400575): 301-311Crossref PubMed Scopus (954) Google Scholar). These membrane-bound structures contained a sundry mix of cellular components, including ribosomes, mitochondria, lipids, and cytosolic enzymes, suggesting that they were signs of autophagy; the researchers therefore named them autophagic bodies (Fig. 2). Noda, the first author of the 1998 JBC paper, was a graduate student in the Ohsumi laboratory. During peer review of the 1992 paper (6Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.J. Cell Biol. 1992; 119 (OnlinePDF 1400575): 301-311Crossref PubMed Scopus (954) Google Scholar), he says, the referees asked for additional experiments to verify that what Noda and Ohsumi saw under the microscope was indeed autophagy. The extra work paid off. “We clearly showed that cytoplasmic enzymes are incorporated into the isolated vacuole during this process,” Noda says, convincing the referees and securing the paper's publication. The referees' requests also prompted Noda to develop more specific and sensitive methods. “After the experience with the first paper, I realized that we needed a quantitative assay for measuring autophagy activity,” he says. In a follow-up paper (8Noda T. Matsuura A. Wada Y. Ohsumi Y. Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae.Biochem. Biophys. Res. Commun. 1995; 210 (OnlinePDF 7741731): 126-132Crossref PubMed Scopus (295) Google Scholar), Noda and others in the Ohsumi lab developed an assay that measures the activity of a genetically engineered alkaline phosphatase (ALP) that becomes active only when translocated to the yeast vacuole as happens during autophagy. “Having this system in hand, I got interested in studying the regulatory mechanism of autophagy in more detail,” says Noda. At this time, Noda came across a recently published paper from the group of Michael Hall at the University of Basel, Switzerland, reporting that Tor in yeast controlled cellular processes that are typically induced in response to starvation (9Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. TOR controls translation initiation and early G1 progression in yeast.Mol. Biol. Cell. 1996; 7 (OnlinePDF 8741837): 25-42Crossref PubMed Scopus (599) Google Scholar). “As autophagy is induced by starvation, I was eager to use our ALP assay to test the hypothesis that Tor might also regulate autophagy,” says Noda. Noda and Ohsumi grew yeast cells in nutrient-rich conditions in which autophagy is typically repressed. The researchers then added the Tor inhibitor rapamycin to the cell cultures. Using their ALP-based autophagy assay, they detected autophagy in the Tor-inhibited cells, and using light microscopy, they also observed the tell-tale autophagic bodies in the vacuoles (3Noda T. Ohsumi Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast.J. Biol. Chem. 1998; 273 (OnlinePDF 9461583): 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). The authors firmed up these observations with a yeast strain that lacked the WT TOR1 and TOR2 genes. The strain carried a TOR2 gene encoding a temperature-sensitive Tor2 variant that functioned normally at 30 °C but became inactivated at 37 °C. Using this TOR2-mutant strain, the authors found that it exhibits autophagy when grown at 37 °C, confirming that the WT Tor protein represses autophagy. Interestingly, Noda and Ohsumi also found that Tor's effect on autophagy was independent of its known activity in cell cycle progression, suggesting that it plays more multifaceted roles than previously thought. “Our discovery of Tor's role in autophagy revised prevailing views at the time, showing that Tor regulates not only anabolic processes, but also catabolic ones,” says Noda. “This coupling of anabolic and catabolic processes has been a key game changer in the research field during the past decades,” he notes. They next turned to the question of how Tor might control autophagy. The Ohsumi lab had previously generated 14 autophagy-deficient (apg) yeast strains (10Tsukada M. Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.FEBS Lett. 1993; 333 (OnlinePDF 8224160): 169-174Crossref PubMed Scopus (1396) Google Scholar), and Noda found that rapamycin did not restore autophagy in these mutants, indicating that the APG genes (now called ATG) all act downstream of Tor (3Noda T. Ohsumi Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast.J. Biol. Chem. 1998; 273 (OnlinePDF 9461583): 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). The Classic paper could not resolve the question how Tor might regulate the ATGs, but there was a promising hint—Tor was known to have a kinase domain essential for cell cycle regulation (11Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression.Cell. 1993; 73 (OnlinePDF 8387896): 585-596Abstract Full Text PDF PubMed Scopus (728) Google Scholar). Although it was not definitively known whether this domain had actual kinase activity (3Noda T. Ohsumi Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast.J. Biol. Chem. 1998; 273 (OnlinePDF 9461583): 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar), its presence suggested that Tor might control autophagy by phosphorylating and thereby inhibiting autophagy-associated proteins such as the ATGs. This was borne out by a 2000 study published by the Ohsumi lab, reporting that Tor phosphorylates the autophagy-related yeast protein Atg13 (12Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. Tor-mediated induction of autophagy via an Apg1 protein kinase complex.J. Cell Biol. 2000; 150 (OnlinePDF 10995454): 1507-1513Crossref PubMed Scopus (907) Google Scholar). This phosphorylation interfered with the ability of Atg13 to bind to and activate another kinase, Atg1, required for autophagy induction. Subsequent work by the Ohsumi lab with the facile yeast system helped pinpoint many other key players in autophagy (13Nakatogawa H. Suzuki K. Kamada Y. Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast.Nat. Rev. Mol. Cell Biol. 2009; 10 (OnlinePDF 19491929): 458-467Crossref PubMed Scopus (1270) Google Scholar). Despite great strides in clarifying autophagy mechanisms in both yeast and mammalian cells (which use the mTOR protein to suppress autophagy (1Thorburn A. Autophagy and disease.J. Biol. Chem. 2018; 293 (OnlinePDF 29191833): 5425-5430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar)), more work is needed. In particular, to prevent self-destruction, “cells must avoid excessive degradation during autophagy,” Noda says, meaning they need mechanisms that terminate autophagy at the right time. However, the details of these proposed mechanisms remain to be worked out. “We are currently investigating this question and already got some good candidate molecules [that might terminate autophagy],” says Noda. For his work that helped uncover the molecular mechanisms in autophagy, Ohsumi was awarded the Nobel Prize in Physiology or Medicine in 2016.

Insulin and glucagon are well-known peptide hormones that keep glucose levels within a healthy range in the body. But they are only part of a complex network that controls concentrations of this ubiquitous sugar in blood and tissues. Other molecules regulate glucose by controlling insulin secretion from the pancreas or protecting pancreatic β cells against stresses that lead to cellular dysfunction or cell death (1).

The discovery in 1959 that metformin (dimethylbiguanide) lowers blood glucose levels in people with type 2 diabetes (1) has been a major boon for millions with this common metabolic disorder. Metformin is currently also being studied as a therapeutic agent for managing other serious maladies, such as fatty liver disease, cardiovascular disorders, and cancer.

Lipids are ubiquitous molecules, serving as the structural building blocks for membranes, messengers in cell signaling, or compounds for energy storage. Their functional versatility relies in part on the presence of one or more double bonds in the long lipid carbon chains. The more double bonds there are in a lipid, the more unsaturated it is; and the more unsaturated lipids there are in a membrane, the more flexible the membrane tends to be. Cells take advantage of this property by fine-tuning the levels of saturated and unsaturated fatty acids in membranes to external conditions and required membrane function. Many organisms, including humans, produce unsaturated fatty acids by desaturating saturated fatty acids, as Rudolf Schoenheimer and David Rittenberg reported in the 1930s working with mammalian cells (1Schoenheimer R. Rittenberg D. Deuterium as an indicator in the study of intermediary metabolism: V. The desaturation of fatty acids in the organism.J. Biol. Chem. 1936; 113 (http://www.jbc.org/content/113/2/505.full.pdf?sid=02ec67ce-5684–45c2-bba4-f47412e5c0b0): 505-510Abstract Full Text PDF Google Scholar) and Konrad Bloch and Daniel Bloomfield showed in the 1960s studying yeast (2Bloomfield D.K. Bloch K. The formation of Δ9-unsaturated fatty acids.J. Biol. Chem. 1960; 235 (http://www.jbc.org/content/235/2/337.full.pdf 13801633): 337-345Abstract Full Text PDF PubMed Google Scholar). When it comes to making unsaturated fatty acids, cells growing in the presence of molecular oxygen have an advantage; they can use this powerful oxidant to insert a double bond into a fatty acid chain via a dehydrogenation reaction. But what about organisms that cannot grow in the presence of molecular oxygen? This question piqued the curiosity of Bloch, who had a longstanding interest in oxygen as a biosynthetic reagent and was also aware that molecular oxygen is toxic to some microorganisms. In the 1950s, he had taken a summer course taught by the eminent microbiologist Cornelius van Niel. The course acquainted Bloch with many microorganisms, including obligately anaerobic bacteria that thrive only in the absence of oxygen (3Bloch K. Summing up.Annu. Rev. Biochem. 1987; 56 (https://www.annualreviews.org/doi/abs/10.1146/annurev.bi.56.070187.000245 3304130): 1-19Crossref PubMed Google Scholar). Howard Goldfine worked with Bloch on bacterial fatty acid biosynthesis and is now emeritus professor of microbiology at the University of Pennsylvania. “[The course] caused him to wonder what goes on in anaerobes, because obviously, they could not use molecular oxygen [for making unsaturated fatty acids],” Goldfine says. Bloch sought to answer this question by studying fatty acid biosynthesis in two species of anaerobic bacteria, Clostridium kluyveri and Clostridium butyricum (strain 6015, now known as Clostridium beijerinckii). “Clostridia are present anywhere where there's no oxygen,” Goldfine says. “If you look in mud several inches below the surface, you'll find clostridia, and they're happily growing.” The work of Bloch, Goldfine, and colleagues on unsaturated fatty acid biosynthesis in clostridia resulted in two JBC papers, recognized as Classics here (4Goldfine H. Bloch K. On the origin of unsaturated fatty acids in clostridia.J. Biol. Chem. 1961; 236 (http://www.jbc.org/content/236/10/2596.full.pdf?sid=2023ec0f-bafb-4973-acd9-e69b436b85d8 13899759): 2596-2601Abstract Full Text PDF PubMed Google Scholar, 5Scheuerbrandt G. Goldfine H. Baronowsky P.E. Bloch K. A novel mechanism for the biosynthesis of unsaturated fatty acids.J. Biol. Chem. 1961; 236 (http://www.jbc.org/content/236/10/PC70.full.pdf?sid=2023ec0f-bafb-4973-acd9-e69b436b85d8 14498314): PC70-PC71Abstract Full Text PDF PubMed Google Scholar). The studies reported that C. kluyveri and C. butyricum 6015 (C. beijerinckii) produce unsaturated fatty acids via a biosynthetic route distinct from that present in aerobic cells. Goldfine's association with Bloch began through their shared interest in anaerobic metabolism. Goldfine had worked on anaerobic bacteria in Earl Stadtman's lab at the National Heart Institute of the National Institutes of Health, prompting Bloch to explore whether Goldfine might be interested in working with him. “He was coming down to NIH for a study section, and he asked if we could have lunch together, and then he asked, would I be interested in coming to visit his lab?” Goldfine remembers. Goldfine was familiar with Bloch's work on cholesterol and fatty acids, for which Bloch won the Nobel prize in physiology and medicine with Feodor Lynen in 1964 (6Kresge N. Simoni R.D. Hill R.L. The biosynthetic pathway for cholesterol: Konrad Bloch.J. Biol. Chem. 2005; 280 (http://www.jbc.org/content/280/10/e7.full?sid=17233771–608b-44fe-abfe-6b1b683e5253): e7Abstract Full Text Full Text PDF Google Scholar). So Goldfine enthusiastically jumped at the opportunity, resulting in a 3-year stay in the Bloch laboratory. “I was really very, very fortunate to have had the opportunity to work with Bloch, because he was a man of really great depth and understanding,” Goldfine says. “It was a turning point in my career.” Goldfine's expertise was soon tested: removing all oxygen from the experimental system to study unsaturated fatty acid formation in clostridia posed a challenge all its own. Initially, Goldfine tried to grow the bacteria in a helium atmosphere in a vacuum desiccator, “but that did not completely remove oxygen, because there could be a trace of oxygen in the helium,” he recalls. So Goldfine resorted to a chemical trick and skillful lab acrobatics. He poured a 5% potassium carbonate solution into the desiccator base, above which he placed the flasks holding the bacteria in growth media, along with a beaker containing pyrogallic acid. After sealing and helium-purging the desiccator, Goldfine gingerly tilted it to tip the pyrogallol into the potassium carbonate solution. The resulting mixture then removed any traces of oxygen from the helium-filled desiccator. Tools such as mass spectrometry to analyze the bacterial lipids were not readily available at the time. Instead, the researchers relied on Bloch's longstanding expertise in using 14C labeling and detection. After feeding the 14C-labeled lipid precursor acetate to the bacterial cultures, Bloch and Goldfine extracted the lipids from the cells, used gas chromatography to separate fatty acids, identified them with standards, and assessed them for 14C labeling in a scintillation counter. In the first study (4Goldfine H. Bloch K. On the origin of unsaturated fatty acids in clostridia.J. Biol. Chem. 1961; 236 (http://www.jbc.org/content/236/10/2596.full.pdf?sid=2023ec0f-bafb-4973-acd9-e69b436b85d8 13899759): 2596-2601Abstract Full Text PDF PubMed Google Scholar), Goldfine and Bloch reported incorporation of two-thirds of the 14C-labeled acetate into saturated lipids and one-third into monounsaturated lipids, showing that bacteria can produce unsaturated fatty acids in the absence of molecular oxygen. This finding raised the question how the clostridia could create double bonds in fatty acids. One possibility was that the bacterial cells possessed the means to desaturate a bond in an existing saturated fatty acid, as previously found in other organisms, but by using an oxidant other than molecular oxygen. Or the clostridia might deploy a different, yet unknown mechanism to generate a lipid double bond. To test these possibilities, Goldfine and Bloch fed a series of 14C-labeled saturated fatty acids ranging in length from 8 to 18 carbons to the cells. They found that the clostridia do not make unsaturated fatty acids by desaturating bonds in their saturated counterparts. Instead, they produce a single double bond during elongation of the saturated fatty acids octanoic acid (C8) and decanoic acid (C10), resulting in long-chain (C12 to C18) monounsaturated fatty acids. Interestingly, the clostridia did not use saturated fatty acid chains longer than 10 carbons for making unsaturated fatty acids. This indicated that these bacteria use short- and medium-chain fatty acids (up to 8 or 10 carbons long) as precursors of both saturated and unsaturated fatty acids through the same biochemical route, but make longer-chain saturated and unsaturated fatty acids via two distinct pathways. Peering into the clostridial lipid products a little more, Bloch and colleagues saw something odd. “C. beijerinckii [presented] an additional problem because the unsaturated fatty acids have two isomers,” says Goldfine. The unsaturated fatty acid hexadecenoic acid isomers had a double bond either at the C7 or C9 (Δ7 or Δ9) position, and octadecenoic acid isomers either at C9 or C11 (Δ9 or Δ11). To find out how these isomers are made, and as described in the second JBC paper (5Scheuerbrandt G. Goldfine H. Baronowsky P.E. Bloch K. A novel mechanism for the biosynthesis of unsaturated fatty acids.J. Biol. Chem. 1961; 236 (http://www.jbc.org/content/236/10/PC70.full.pdf?sid=2023ec0f-bafb-4973-acd9-e69b436b85d8 14498314): PC70-PC71Abstract Full Text PDF PubMed Google Scholar), Bloch, Goldfine, and another member of the Bloch lab, Günter Scheuerbrandt, fed 14C-labeled octanoic and decanoic acids to C. beijerinckii and again measured 14C incorporation into hexadecenoic and octadecenoic acids. They tweaked their procedure to include exposure of the unsaturated fatty acids to potassium metaperiodate-potassium permanganate (KIO4–KMnO4), a strong oxidant that breaks lipid double bonds. This enabled them to trace the origins of both ends of the unsaturated products and home in on the mechanism that creates the different fatty acid isomers. “I distinctly remember, Paul Baronowsky—he was a graduate student at the time—standing at the blackboard at the end of the lab writing that pathway,” Goldfine says. “'This is how you get the double bond here. And if you do it with another precursor, you get the double bond there.’ He drew it all out beautifully.” Their carefully designed experiments and analyses revealed that the individual isomers do not arise from interconversions among them. Instead, the researchers found that octanoic acid is the precursor to the long-chain fatty acids having a double bond at the C9 or C11 position and decanoic acid is the precursor to those with a double bond at the C7 or C9 position (Fig. 1). The authors' findings supported an earlier proposal by famed chemist Klaus Hofmann at the University of Pittsburgh. Working with lactobacilli, Hofmann and colleagues had speculated that bacteria may produce long-chain unsaturated fatty acids by chain elongation from a short-chain precursor (7Hofmann K. O'Leary W.M. Yoho C.W. Liu T.-Y. Further observations on lipide stimulation of bacterial growth.J. Biol. Chem. 1959; 234 (http://www.jbc.org/content/234/7/1672.full.pdf 13672943): 1672-1677Abstract Full Text PDF PubMed Google Scholar). On the basis of their published and unpublished findings, Bloch and colleagues proposed that in clostridia and several other bacteria, including lactobacilli and Escherichia coli, the long-chain unsaturated fatty acids are produced by an anaerobic pathway involving elongation of octanoic acid or decanoic acid, immediately followed by a β,γ elimination of water from a hydroxy acid intermediate and further elongation of the lipid chain without reduction of the double bond (5Scheuerbrandt G. Goldfine H. Baronowsky P.E. Bloch K. A novel mechanism for the biosynthesis of unsaturated fatty acids.J. Biol. Chem. 1961; 236 (http://www.jbc.org/content/236/10/PC70.full.pdf?sid=2023ec0f-bafb-4973-acd9-e69b436b85d8 14498314): PC70-PC71Abstract Full Text PDF PubMed Google Scholar). Further studies in the Bloch lab, done with E. coli (8Lennarz W. Light R. Bloch K. A fatty acid synthetase from E. coli.Proc. Natl. Acad. Sci. U.S.A. 1962; 48 (16590950): 840-846https://www.ncbi.nlm.nih.gov/pmc/articles/PMC220863/Crossref PubMed Google Scholar, 9Norris A.T. Bloch K. On the mechanism of the enzymatic synthesis of unsaturated fatty acids in Escherichia coli.J. Biol. Chem. 1963; 238 (http://www.jbc.org/content/238/9/PC3133.full.pdf 14081939): PC3133Abstract Full Text PDF Google Scholar10Norris A.T. Matsumura S. Bloch K. Fatty acid synthetase and β-hydroxydecanoyl coenzyme A dehydrase from Escherichia coli.J. Biol. Chem. 1964; 239 (http://www.jbc.org/content/239/11/3653.full.pdf 14257590): 3653-3662Abstract Full Text PDF PubMed Google Scholar), focused on the bacterial enzymes involved in fatty acid chain elongation and double bond formation. This work culminated in two papers confirming that the anaerobic bacterial pathway indeed involves enzymatic dehydration of medium-chain β-hydroxy acids rather than oxidative dehydrogenation of a saturated acid (11Kass L.R. Brock D.J.H. Bloch K. β-Hydroxydecanoyl thioester dehydrase I. Purification and properties.J. Biol. Chem. 1967; 242 (http://www.jbc.org/content/242/19/4418.long 4863739): 4418-4431Abstract Full Text PDF PubMed Google Scholar, 12Brock D.J.H. Kass L.R. Bloch K. β-Hydroxydecanoyl thioester dehydrase II. Mode of action.J. Biol. Chem. 1967; 242 (http://www.jbc.org/content/242/19/4432.long 4863740): 4432-4440Abstract Full Text PDF PubMed Google Scholar). Thus, innovative rejiggering of lab equipment, foundational biochemistry methods, and several incisive blackboard sketches yielded results that uncovered how anaerobes make unsaturated fatty acids and that have stood the test of time to this day. “The pathway that is in the second JBC [Classics] paper has held up for 60 years, it's pretty astonishing,” concludes Goldfine.

Some scientific discoveries land with a boom only to fizzle out and become a small blip—but there are times when this order is reversed. Such was the case with the discovery of γ-aminobutyric acid (GABA) in the brain, reported in 1950. In a study published in the Journal of Biological Chemistry (1), preceded by a brief conference report shortly before that (2), Eugene Roberts (Fig. 1) and Sam Frankel not only identified GABA as a major amine in the brain, but also reported that it is produced and preferentially accumulates in this organ.

The human body regularly encounters and combats many pathogenic organisms and toxic molecules. Its ensuing responses to these disease-causing agents involve two interrelated systems: innate immunity and adaptive (or acquired) immunity. Innate immunity is active at several levels, both at potential points of entry and inside the body (see figure). For example, the skin represents a physical barrier preventing pathogens from invading internal tissues. Digestive enzymes destroy microbes that enter the stomach with food. Macrophages and lymphocytes, equipped with molecular detectors, such as Toll-like receptors (TLRs), which latch onto foreign structures and activate cellular defenses, patrol the inside of the body. These immune cells sense and devour microbes, damaged cells, and other foreign materials in the body. Certain proteins in the blood (such as proteins of the complement system and those released by natural killer cells, along with antimicrobial host-defense peptides) attach to foreign organisms and toxins to initiate their destruction.

Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed.

Genetic nonself recognition systems, termed heterokaryon or vegetative incompatibility (vic), operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals. The vic system operating in the chestnut blight fungus, Cryphonectria parasitica, has been reported to restrict transmission of virulence-attenuating mycoviruses. A comparative genomics approach has been employed to identify physical genes associated with the six genetically identified C. parasitica vic loci.

An inducible RNA-silencing pathway, involving a single Dicer protein, DCL2, and a single Argonaute protein, AGL2, was recently shown to serve as an effective antiviral defense response in the chestnut blight fungus Cryphonectria parasitica. Eukaryotic RNA-dependent RNA polymerases (RdRPs) are frequently involved in transcriptional and posttranscriptional gene silencing and antiviral defense. We report here the identification and characterization of four RdRP genes (rdr1-4) in the C. parasitica genome. Sequence relationships with other eukaryotic RdRPs indicated that RDR1 and RDR2 were closely related to QDE-1, an RdRP involved in RNA silencing ("quelling") in Neurospora crassa, whereas RDR3 was more closely related to the meiotic silencing gene SAD-1 in N. crassa. The RdRP domain of RDR4, related to N. crassa RRP-3 of unknown function, was truncated and showed evidence of alternative splicing. Similar to reports for dcl2 and agl2, the expression levels for rdr3 and rdr4 increased after hypovirus CHV-1/EP713 infection, while expression levels of rdr1 and rdr2 were unchanged. The virus-responsive induction patterns for rdr3 and rdr4 were altered in the Δdcl2 and Δagl2 strains, suggesting some level of interaction between rdr3 and rdr4 and the dcl2/agl2 silencing pathway. Single rdr gene knockouts Δrdr1-4, double knockouts Δrdr1/2, Δrdr2/3, Δrdr1/3, and a triple knockout, Δrdr1/2/3, were generated and evaluated for effects on fungal phenotype, the antiviral defense response, viral RNA recombination activity and transposon expression. None of the single or multiple rdr knockout strains displayed any phenotypic differences from the parental strains with or without viral infection or any significant changes in viral RNA accumulation or recombination activity or transposon RNA accumulation, indicating no detectable contribution by the C. parasitica rdr genes to these processes.

The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

ABSTRACT Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. ΔΔsfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔsfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔsfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.