TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Microbial liberation of N-methylserotonin from orange fiber in gnotobiotic mice and humans.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

Dicer-like Enzymes with Sequence Cleavage Preferences.

Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins, which select the guide strand, often with a 5' end sequence bias. However, Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development, two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2, Dcl3, and Dcl5. Through in vitro cleavage assays, we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and 5' AGA, while Dcl3 has a sequence preference for 5' UNG. Dcl5, however, has cleavage preferences for 5' UAG and 3' CUAC/UN, which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus, we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.

At the Intersection of Chemistry, Biology, and Medicine.

After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 105 to 106 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.

Lipopolysaccharide-binding protein expression is increased by stress and inhibits monoamine synthesis to promote depressive symptoms.

Monoamine insufficiency is suggested to be associated with depressive features such as sadness, anhedonia, insomnia, and cognitive dysfunction, but the mechanisms that cause it are unclear. We found that the acute-phase protein lipopolysaccharide-binding protein (LBP) inhibits monoamine biosynthesis by acting as an endogenous inhibitor of dopamine-ß-hydroxylase (DBH) and aromatic-L-amino-acid-decarboxylase (DDC). LBP expression was increased in individuals with depression and by diverse stress challenges in mice. LBP antibodies and LBP knockdown inhibited monoamine insufficiency and depression-like features in mice, which worsened with LBP overexpression or administration. Monoamine insufficiency and depression-like symptoms were not induced by stressful stimuli in LBP-deficient mice, further highlighting a role for LBP in stress-induced depression, and a peptide we designed that blocks LBP-DBH and LBP-DDC interactions showed anti-depression effects in mice. This study reveals an important role for LBP in regulating monoamine biosynthesis and suggests that targeting LBP may have potential as a treatment for some individuals with depression.

RNA Binding to CBP Stimulates Histone Acetylation and Transcription.

CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP-a regulatory motif unique to CBP/p300-allows RNA to stimulate CBP's HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes.

Assessing the Contributions of Motor Enzymes and Microtubule Dynamics to Mitotic Chromosome Motions.

During my graduate work with Keith Porter, I became fascinated by the mitotic spindle, an interest that has motivated much of my scientific work ever since. I began spindle studies by using electron microscopes, instruments that have made significant contributions to our understanding of spindle organization. Such instruments have helped to elucidate the distributions of spindle microtubules, the interactions among them, their molecular polarity, and their associations with both kinetochores and spindle poles. Our lab has also investigated some processes of spindle physiology: microtubule dynamics, the actions of microtubule-associated proteins (including motor enzymes), the character of forces generated by specific spindle components, and factors that control mitotic progression. Here, I give a personal perspective on some of this intellectual history and on what recent discoveries imply about the mechanisms of chromosome motion.

Cellulose degradation by polysaccharide monooxygenases.

Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity.

Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs.

Over the last two decades, hydrogen sulfide (H2S) has emerged as an endogenous regulator of a broad range of physiological functions. H2S belongs to the class of molecules known as gasotransmitters, which typically include nitric oxide (NO) and carbon monoxide (CO). Three enzymes are recognized as endogenous sources of H2S in various cells and tissues: cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). The present article reviews the regulation of these enzymes as well as the pathways of their enzymatic and nonenzymatic degradation and elimination. The multiple interactions of H2S with other labile endogenous molecules (e.g., NO) and reactive oxygen species are also outlined. Next, the various biological targets and signaling pathways are outlined, with special reference to H2S or oxidative posttranscriptional modification (persulfidation or sulfhydration) of proteins and the effect of H2S on various channels and intracellular second messenger pathways, the regulation of gene transcription and translation, and the regulation of cellular bioenergetics and metabolism. The pharmacological and molecular tools currently available to study H2S physiology are also reviewed, including their utility and limitations. In subsequent sections, the role of H2S in the regulation of various physiological and cellular functions is reviewed, including the regulation of membrane potential, endo- and exocytosis, regulation of various cell organelles (endoplasmic reticulum, Golgi, mitochondria), regulation of cell movement, cell cycle, cell differentiation, and physiological aspects of regulated cell death. Next, the physiological roles of H2S in various cell types and organ systems are overviewed, including the role of H2S in red blood cells, immune cells, the central and peripheral nervous systems (with focus on neuronal transmission, learning, and memory formation), and regulation of vascular function (including angiogenesis as well as its specialized roles in the cerebrovascular, renal, and pulmonary vascular beds) and the role of H2S in the regulation of special senses, vision, hearing, taste and smell, and pain-sensing. Finally, the roles of H2S in the regulation of various organ functions (lung, heart, liver, kidney, urogenital organs, reproductive system, bone and cartilage, skeletal muscle, and endocrine organs) are presented, with a focus on physiology (including physiological aging) but also extending to some common pathophysiological conditions. From these data, a wide array of significant roles of H2S in the physiological regulation of all organ functions emerges and the characteristic bell-shaped biphasic effects of H2S are highlighted. In addition, key pathophysiological aspects, debated areas, and future research and translational areas are identified.

Captured snapshots of PARP1 in the active state reveal the mechanics of PARP1 allostery.

PARP1 rapidly detects DNA strand break damage and allosterically signals break detection to the PARP1 catalytic domain to activate poly(ADP-ribose) production from NAD+. PARP1 activation is characterized by dynamic changes in the structure of a regulatory helical domain (HD); yet, there are limited insights into the specific contributions that the HD makes to PARP1 allostery. Here, we have determined crystal structures of PARP1 in isolated active states that display specific HD conformations. These captured snapshots and biochemical analysis illustrate HD contributions to PARP1 multi-domain and high-affinity interaction with DNA damage, provide novel insights into the mechanics of PARP1 allostery, and indicate how HD active conformations correspond to alterations in the catalytic region that reveal the active site to NAD+. Our work deepens the understanding of PARP1 catalytic activation, the dynamics of the binding site of PARP inhibitor compounds, and the mechanisms regulating PARP1 retention on DNA damage.

Sortases: structure, mechanism, and implications for protein engineering.

Sortase enzymes are critical cysteine transpeptidases on the surface of bacteria that attach proteins to the cell wall and are involved in the construction of bacterial pili. Due to their ability to recognize specific substrates and covalently ligate a range of reaction partners, sortases are widely used in protein engineering applications via sortase-mediated ligation (SML) strategies. In this review, we discuss recent structural studies elucidating key aspects of sortase specificity and the catalytic mechanism. We also highlight select recent applications of SML, including examples where fundamental studies of sortase structure and function have informed the continued development of these enzymes as tools for protein engineering.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Directing RNA-modifying machineries towards endogenous RNAs: opportunities and challenges.

Over 170 chemical modifications can be naturally installed on RNA, all of which are catalyzed by dedicated machineries. These modifications can alter RNA sequence structure, stability, and translation as well as serving as quality control marks that record aspects of RNA processing. The diverse roles played by RNAs within cells has motivated endeavors to exogenously introduce RNA modifications at target sites for diverse purposes ranging from recording RNA:protein interactions to therapeutic applications. Here, we discuss these applications and the approaches that have been employed to engineer RNA-modifying machineries, and highlight persisting challenges and perspectives.

Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Blocking endogenous retinoic acid degradation induces oral tooth formation in zebrafish.

According to Dollo's Law of irreversibility in evolution, a lost structure is usually considered to be unable to reappear in evolution due to the accumulation over time of mutations in the genes required for its formation. Cypriniform fish are a classic model of evolutionary loss because, while they form fully operational teeth in the ventral posterior pharynx, unlike other teleosts, they do not possess oral teeth. Paleontological data show that Cypriniforms, a clade of teleost fish that includes the zebrafish, lost their oral teeth 50 to 100 Mya. In order to attempt to reverse oral tooth loss in zebrafish, we block the degradation of endogenous levels of retinoic acid (RA) using a specific inhibitor of the Cyp26 RA degrading enzymes. We demonstrate the inhibition of endogenous RA degradation is sufficient to restore oral tooth induction as marked by the re-appearance of expression of early dental mesenchyme and epithelium genes such as dlx2b and sp7 in the oral cavity. Furthermore, we show that these exogenously induced oral tooth germs are able to be at least partly calcified. Taken together, our data show that modifications of signaling pathways can have a significant effect on the reemergence of once-lost structures leading to experimentally induced reversibility of evolutionary tooth loss in cypriniforms.

Proteasome substrate receptors and their therapeutic potential.

The ubiquitin-proteasome system (UPS) is critical for protein quality control and regulating protein lifespans. Following ubiquitination, UPS substrates bind multidomain receptors that, in addition to ubiquitin-binding sites, contain functional domains that bind to deubiquitinating enzymes (DUBs) or the E3 ligase E6AP/UBE3A. We provide an overview of the proteasome, focusing on its receptors and DUBs. We highlight the key role of dynamics and importance of the substrate receptors having domains for both binding and processing ubiquitin chains. The UPS is rich with therapeutic opportunities, with proteasome inhibitors used clinically and ongoing development of small molecule proteolysis targeting chimeras (PROTACs) for the degradation of disease-associated proteins. We discuss the therapeutic potential of proteasome receptors, including hRpn13, for which PROTACs have been developed.

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase.

Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2's cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.

USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

N6-Methyladenosines Modulate A-to-I RNA Editing.

N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) editing are two of the most abundant RNA modifications, both at adenosines. Yet, the interaction of these two types of adenosine modifications is largely unknown. Here we show a global A-to-I difference between m6A-positive and m6A-negative RNA populations. Both the presence and extent of A-to-I sites in m6A-negative RNA transcripts suggest a negative correlation between m6A and A-to-I. Suppression of m6A-catalyzing enzymes results in global A-to-I RNA editing changes. Further depletion of m6A modification increases the association of m6A-depleted transcripts with adenosine deaminase acting on RNA (ADAR) enzymes, resulting in upregulated A-to-I editing on the same m6A-depleted transcripts. Collectively, the effect of m6A on A-to-I suggests a previously underappreciated interplay between two distinct and abundant RNA modifications, highlighting a complex epitranscriptomic landscape.

Facile and dynamic cleavage of every iron-sulfide bond in cuboidal iron-sulfur clusters.

Nature employs weak-field metalloclusters to support a wide range of biological processes. The most ubiquitous metalloclusters are the cuboidal Fe-S clusters, which are comprised of Fe sites with locally high-spin electronic configurations. Such configurations enhance rates of ligand exchange and imbue the clusters with a degree of structural plasticity that is increasingly thought to be functionally relevant. Here, we examine this phenomenon using isotope tracing experiments. Specifically, we demonstrate that synthetic [Fe4S4] and [MoFe3S4] clusters exchange their Fe atoms with Fe2+ ions dissolved in solution, a process that involves the reversible cleavage and reformation of every Fe-S bond in the cluster core. This exchange is facile-in most cases occurring at room temperature on the timescale of minutes-and documented over a range of cluster core oxidation states and terminal ligation patterns. In addition to suggesting a highly dynamic picture of cluster structure, these results provide a method for isotopically labeling pre-formed clusters with spin-active nuclei, such as 57Fe. Such a protocol is demonstrated for the radical S-adenosyl-l-methionine enzyme, RlmN.