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Receptors for Antigen Uptake
No full textSlide 4-9: Receptors for antigen uptake, processing, and presentation on MHC class I and II productshttps://digitalcommons.rockefeller.edu/endocytosis/1008/thumbnail.jp
Exogenous Pathway
No full textSlide 4-8: Exogenous pathway for MHC I - peptide complexes from dying cells and immune complexeshttps://digitalcommons.rockefeller.edu/endocytosis/1007/thumbnail.jp
Tumor/GalCer
No full textSlide 5-27https://digitalcommons.rockefeller.edu/nkt-cells/1025/thumbnail.jp
The Need to Mature Dendritic Cells
No full textSlide 5-36https://digitalcommons.rockefeller.edu/nkt-cells/1034/thumbnail.jp
Antigen From EL4/GalCer
No full textSlide 5-32https://digitalcommons.rockefeller.edu/nkt-cells/1030/thumbnail.jp
Merz Trio
No full textMerz Trio, piano trio: Brigid Coleridge, violin; Julia Yang, cello; Lee Dionne, piano, performed Haydn: Piano Trio No. 44 in E Major, Hob. XV/28; Alban Berg: Sieben Frühe Lieder (Seven Early Songs) - IV. Traumgekrönt; Ludwig van Beethoven: Piano Trio No. 7 in B-flat Major, Op. 97 “Archduke.
Photo credit: Walter W. Naumburg Foundation
Merz Trio -- Beethoven Op. 70 No. 2: Finalehttps://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1007/thumbnail.jp
Mechanisms of Evasion: Anti-Tumor Immune Suppression & TRNA-Dependent Growth in Cancer Metastasis
Get PDFMetastatic disease presents diverse therapeutic challenges for cancer patients, clinicians, and scientists. Cancer cells have developed sophisticated mechanisms of evasion to survive the selective pressures provided by the metastatic cascade. Two themes of success encompass modulating their surrounding microenvironment, or manipulating cell autonomous regulatory programs not intended for differentiated somatic cells. Both challenges are addressed in this body of work which focus on gastrointestinal and breast cancer metastasis. During cancer progression, tumour cells employ mechanisms that suppress both adaptive and innate immune responses. While our understanding of the molecular basis of adaptive immune suppression by cancer cells has led to the development of transformative immune checkpoint therapies, our knowledge of the molecular basis of innate immune suppression is less developed. Such innate immune suppressive mechanisms are thought to be especially active in gastrointestinal cancers— prevalent malignancies that are overall highly refractory to approved immune therapies. We describe an elegant molecular mechanism employed by pancreatic and colorectal cancer cells for repression of innate anti-tumour immunity. We observe that the creatine kinase brain-type (CKB) enzyme, which is over-expressed by colorectal cancer cells suppresses anti-tumor immunity. CKB localizes to the surface of cancer cells and can also be released into the extracellular space. This kinase enzymatically consumes extracellular ATP—a potent immune stimulatory Danger Associated Molecular Pattern (DAMP) molecule—from the tumor microenvironment. ATP hydrolysis by CKB generates ADP, an immune suppressive molecule. The action of this enzyme can thus concomitantly deplete an immune stimulatory molecule and generate an immune suppressive metabolite. Consistent with this, tumoral CKB suppresses dendritic cell activation, T-cell activation and B cell infiltration. Moreover, CKB suppresses immunity against primary and metastatic pancreatic tumors and impairs establishment of immunological antitumoral memory. To uncover new modalities of cell autonomous regulation by cancer cells, in the midst of technical development to measure small RNA species, tRNAs have emerged as significant regulators of gene expression. The human genome contains 61 codons that are recognized by distinct transfer RNAs (tRNAs). We report the surprising observation that two isoacceptor tRNAs that decode synonymous codons become modulated in opposing directions during breast cancer progression. Specifically, tRNAIle UAU is upregulated whereas tRNAIle GAU is repressed as breast cancer cells attain enhanced metastatic capacity. TRNAIle UAU promoted and tRNAIle GAU suppressed metastatic colonization. These effects were mediated by codon-dependent translation of growth promoting or suppressing genes via cognate codon-dependent interactions. We also make the surprising observation that one isoleucyl tRNA can competitively impair translational decoding of synonymous codons by the other tRNA. Our findings uncover a specific isoacceptor tRNA pair that act in opposition—modulating distinct gene networks that contribute to an organismal phenotype. The degeneracy of the genetic code can thus be biologically exploited by human cancer cells via tRNA isoacceptor shifts and tRNA-mediated translational antagonism at the ribosome. Investigating fundamental cellular programs and tumor microenvironment interactions will enable more opportunity through knowledge to therapeutically target metastatic disease
Cryo-EM Studies of Bacterial RNA Polymerase
Get PDFIn bacteria, a single RNA polymerase (RNAP) performs all transcription. The overall structure of bacterial RNAP resembles a crab claw with pincers comprising the β\u27 and β subunits and a large cleft where the active site sits. Structural information about this essential enzyme has mainly been provided by X-ray crystal structures of stable transcription complexes. RNAP crystal structures are difficult to obtain and the captured states may not always represent physiological states of the enzyme due to crystal packing artifacts. Recent advances in electron detectors and software enable near-atomic resolution structures of large biological complexes to be determined by single particle cryo-electron microscopy (cryo-EM). Unlike X-ray crystallography, cryo-EM samples can be directly visualized without crystallization. In this thesis, I optimized and utilized cryo-EM methodologies to structurally characterize several bacterial RNAP complexes from Escherichia coli (Eco) and Mycobacterium tuberculosis (Mtb): (1) Eco σ70-holoenzyme (Eσ70) in complex with the Eco non-coding RNA (ncRNA) 6S RNA; (2) Mtb RNAP bound to the RNAP inhibitor Fidaxomicin (Fdx); (3) Eσ70 bound to the F element-encoded TraR protein; and (4) Eσ70-dependent promoter DNA melting intermediates stabilized by the TraR transcription factor. Using cryo-EM, I captured RNAP structures that were intractable to crystallization, visualized multiple RNAP conformational states populated in solution, deconvoluted RNAP molecular motions, and observed transient complexes. The work in this thesis showcases the power of cryo-EM to examine macromolecular machines in action. (1) Bacterial 6S RNAs globally regulate transcription by RNAP, directly competing with promoter DNA binding. During transitions between exponential and stationary growth phases, Eco 6S RNA plays a key role in the transcriptional reprogramming by interacting specifically with the housekeeping Eσ70. During my initial cryo-EM experiments with the Eco 6S RNA-Eσ70 complex, I encountered a severe particle orientation bias in my samples. I discovered that the zwitterionic detergent 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) was uniquely effective at solving this issue for bacterial RNAPs. Using this detergent, I determined the cryo-EM structure of the Eco 6S RNA/Eσ70 complex in combination with footprinting and crosslinking approaches in order to elucidate the structural mechanism of 6S RNA mediated inhibition of Eσ70. The structure reveals that 6S RNA is composed of duplex RNA segments that have A-form C3\u27-endo sugar puckers but with widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. The results showed how 6S RNA specifically targets Eσ70 and how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP. (2) Fdx is a RNAP-binding drug that is highly effective against Mtb RNAP in vitro. In collaboration with postdoctoral fellow Hande Boyaci, we solved cryo-EM structures of Mtb σA-holoenzyme (σA-holo) bound to the actinobacteria general transcription factor RbpA and an upstream promoter DNA fork (us-fork) in the presence and absence of Fdx to identify the structural determinants of Fdx binding to RNAP. The results show that Fdx acts like a doorstop fo jam the RNAP clamp module in an open conformation. The structures define the Fdx binding pocket, which includes contacts with RbpA, explaining the drug\u27s strong effect on Mtb. (3) Under starvation conditions, Eco changes the expression of almost one-quarter of its genes within 5 minutes. This global response depends on two factors, the alarmone ppGpp, and the transcription factor DksA. Unlike most transcription factors, ppGpp and DksA bind directly to RNAP but not to DNA. Another transcription factor, TraR, regulates RNAP the same way as ppGpp and DksA. TraR, and ppGpp/DksA, can activate or inhibit transcription initiation depending on the promoter sequence. TraR inhibits Eσ70-dependent transcription from ribosomal RNA and ribosomal protein promoters and activates amino acid biosynthesis and transport promoters in vivo and in vitro. To understand how these factors modulate transcription initiation, I solved cryo-EM structures of Eσ70, with and without TraR, and an open promoter complex (RPo) using a ribosomal promoter that is regulated by TraR, rpsT P2. My cryo-EM structural analyses show that TraR regulates Eσ70 transcription initiation by binding and inducing major conformational changes to mobile RNAP domains that affect RNAP-promoter DNA interactions. (4) Transcription initiation is a multi-step process that leads to the formation of RPo. Using TraR to modulate Eσ70 transcription initiation on the ribosomal protein promoter rpsT P2, I observed promoter DNA melting intermediates by cryo-EM. These structures and supporting biochemical data delineate steps of the RPo formation pathway and provide insight into transient promoter-RNAP interactions that occur along this pathway. These structures span the initial recognition of the duplex promoter in a closed complex (RPc) to the final RPo. Findings from this thesis provide structural and biochemical insight into bacterial RNAP regulation and function. The work in this thesis shows how single-particle cryo-EM can be applied to study transcription factors and small molecules that modulate RNAP as well as transient transcriptional states. This thesis research outlines a framework for future studies of bacterial transcription complexes using cryo-EM
Dole, Vincent P.
No full textVincent Dole, circa 1940s
Courtesy of the Rockefeller Archive Center
Vincent P. Dole (1913-2006) was an American physician, clinician, and professor emeritus of Rockefeller University best known for developing methadone maintenance treatment for heroin addiction with his wife, psychiatrist Marie Nyswander (1919-1986). Born in Chicago in 1913, Dole obtained his undergraduate degree in mathematics from Stanford University in 1934 before studying medicine at Harvard University, receiving his M.D. in 1939. He joined the Rockefeller Institute (which later became the Rockefeller University) in 1941, where his early research focused on the role that sodium and lipids played in metabolism, obesity, and high blood pressure. His work during this time, including a liquid diet that he developed, received professional and commercial attention.
In the early 1960s, Dr. Dole became troubled by the growing drug problem in New York City and decided to devote his laboratory to the study of addiction from a metabolic perspective. He consulted Dr. Marie Nyswander, the only person working in the field at the time who considered drug addiction to be a disease and not a personality defect. During their research in 1964 and 1965, they noticed a significant difference in the ways patients responded to methadone compared to the other substances they were testing. Methadone, a synthetic opioid developed in Germany during World War II, blocked patients\u27 insatiable craving for heroin and restored their self-worth and ability to lead productive lives, as opposed to the extremely low retention rates and high relapse rates of programs that abruptly detoxed patients or attempted to eliminate their socially destructive tendencies, which Dole and Nyswander interpreted as drug-seeking behavior fueled by a disease kept in check by regular doses of methadone, comparable to a diabetic\u27s need for insulin.
Dole and Nyswander received widespread recognition for their work in scientific communities and addiction communities alike. They were the recipients of the first annual Nyswander-Dole award in 1982, sponsored by the New York Urban Coalition, the New York State Division of Substance Abuse, and the Committee of Methadone Program Administrations. Dole also was the recipient of the prestigious Lasker Prize in 1988 and the Prince Mahidol Award in 1996. He was the author of over 100 scientific papers.
Years at The Rockefeller University: 1941-1983; emeritus 1983 -2006https://digitalcommons.rockefeller.edu/faculty-members/1022/thumbnail.jp
Dendritic Cells Capture Cell-Associated Antigens In Vivo
No full textSlide 9-11: Dendritic cells capture cell-associated antigens in vivohttps://digitalcommons.rockefeller.edu/diagrams/1010/thumbnail.jp