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A Proteomic Approach to Elucidating the Function of Picornavirus 2A Protease
Get PDFHuman-infecting viruses have evolved diverse strategies to enter cells and hijack the host machinery to promote their self-replication. Viruses deploy their proteins to subvert a number of host functions, such as the cell cycle, cellular metabolism, protein synthesis, nuclear and RNA transport across the nuclear pore complex, apoptosis, and innate immune responses. Picornaviruses are the most dominant human disease-causing viruses and present an excellent clinical target for research studies into their molecular mechanisms. Picornaviruses have an RNA genome that is translated as a single polyprotein, which is processed into individual components by two proteases, termed 2A and 3C. In addition to polyprotein processing, these proteases also subvert host cell function through cleavage of specific protein targets. The 2A protease is especially critical during the initial stages of picornaviral infections. We developed protein expression platforms to further characterize the 2A protease\u27s function, interacting partners, and targeting mechanisms, showing that it makes surgical strikes against eIF4G and Nup98, key players in host protein synthesis and nucleocytoplasmic trafficking of proteins and RNA, respectively. We subsequently utilized those protease expression platforms in combination with transport reporter assays to interrogate nuclear import and export through the NPC. By studying the interactome of 2A protease, we discovered that it seems to employ two different cleavage mechanisms for its primary targets, Nup98 and eIF4G. It directly binds and degrades Nup98, and may alternatively bind eIF3L and utilize the eIF3 complex as a targeting platform to cleave eIF4G. Cellular fractionation revealed Nup98 cleavage by 2A protease results in observable dissociation of Rae1 from the NPC as well as cytoplasmic accumulation of proteins normally transported by Karyopherins that interact with Nup98 FG motifs. We developed fluorescent transport reporters with various nuclear import and export signals to elucidate their transport mechanism and dependence on Nup98. Nuclear localization signals promiscuously recognized by a variety of Importins were marginally, if at all, affected by Nup98 depletion, while export and import signals depending on Crm1 or Rae1 mediated RNA export were severely affected by the absence of full-length Nup98. We propose a novel cleavage mechanism for the 2A protease, as well as report on our development of a suite of molecular tools to further characterize 2A protease function and nucleocytoplasmic transport mediated by Nup98. These tools can be adapted to include a diverse variety of viral proteins to further characterize host subversion mechanisms as well as other aspects of nuclear pore complex function
Biochemical Studies of Peptidoglycan Hydrolases from Commensal and Pathogenic Bacteria
Get PDFThe intestinal microbiota consists of diverse bacterial species and their effectors that play key roles in regulating human health. Interestingly, cell wall, or peptidoglycan, fragments from commensal and pathogenic bacteria can activate host immunity. The mechanism(s) by which immunologically active peptidoglycan fragments are generated, however, are not well-understood. In this regard, peptidoglycan hydrolases are ubiquitous in bacteria and possess diverse activities to remodel the cell wall during cell growth and division. These peptidoglycan hydrolases can also generate cell wall fragments in this process that are shed or recycled and available for triggering host immunity. In this thesis, we describe methodologies for the biochemical characterization of the NlpC/p60 family of peptidoglycan hydrolases to harness their catalytic activity in multiple therapeutic applications involving commensal bacteria, and to target these proteins in pathogenic bacteria. In Chapter 1, we introduce the NlpC/p60 protein family and review the functions of NlpC/p60 hydrolases in bacterial cell division, their diverse biochemical activities, structurefunction relationships, and novel functions beyond bacterial cell division. In Chapter 2, we describe methods for the biochemical characterization of NlpC/p60 hydrolase activity. These methods encompass bacterial expression and purification of recombinant NlpC/p60 proteins, large scale isolation of peptidoglycan, and in vitro activity assays of NlpC/p60 hydrolase activity. Our optimized methodologies were employed to determine the peptidoglycan substrate specificity of the NlpC/p60 hydrolase SagA from the commensal bacterium Enterococcus faecium. These methods also enabled the evaluation of features identified in the SagA-NlpC/p60 domain structure that may be important for binding peptidoglycan substrates. Moreover, SagA-generated peptidoglycan products can activate a pattern recognition receptor in mammalian cells, which directly links SagA NlpC/p60 hydrolase activity to the activation of host immune pathways, as observed with SagA+ bacteria in mouse models of enteric infection. Our results emphasize the utility of the biochemical analysis of NlpC/p60 hydrolase activity. In Chapter 3, we uncover the localization of SagA in Enterococcus and describe structurefunction studies of the SagA-NlpC/p60 domain. Imaging studies of SagA+ Enterococcus species and fluorescently-tagged SagA constructs implicated SagA in peptidoglycan remodeling in actively dividing Enterococcus cells. Comparative analysis of full-length SagA and SagANlpC/ p60 indicated that the coiled-coil SagA N-terminus may play a scaffolding or targeting function during peptidoglycan remodeling as opposed to an autoinhibitory role. A peptidoglycanbound model of the SagA-NlpC/p60 structure along with alanine screening revealed key ligandprotein interactions that may govern Enterococcus peptidoglycan turnover by SagA-NlpC/p60. From this analysis, we define the catalytic dyad in the NlpC/p60 domain of SagA from multiple commensal enterococci. Together, these results highlight the possible role of SagA in E. faecium viability and provide mechanistic insight into how SagA NlpC/p60 hydrolase activity processes peptidoglycan and generates immunostimulatory cell wall fragments. In Chapter 4, we explore SagA-mediated activation of host immunity and improved responsiveness to anticancer therapy in mouse tumor models. Enterococcus species and strains were recovered from cancer patients demonstrating responsiveness to cancer immunotherapy, but the molecular mechanism(s) behind this association were not understood. Here, we show that Enterococcus species and strains possessing SagA orthologs with highly conserved NlpC/p60 domains specifically enhanced efficacy of cancer immunotherapy by checkpoint blockade. Our optimized biochemical methods were applied to show that the active enterococci share conserved SagA expression, cell wall composition, and in vitro NlpC/p60 hydrolase activity of the respective SagA orthologs. In this analysis, I characterized the peptidoglycan hydrolase activity of a SalA from the non-protective Enterococcus faecalis. Heterologous expression of SagA in E. faecalis showed that SagA is sufficient for improving the antitumor activity of several antibodies. Moreover, using engineered strains of the probiotic Lactococcus lactis that heterologously expressed SagA constructs, we validated that SagA-mediated antitumor activity is dependent on secretion of catalytically active SagA. These results provide further evidence of the therapeutic applicability of SagA NlpC/p60 hydrolase activity. In Chapter 5, we explore targeting NlpC/p60 hydrolase activity in the context of multi-drug resistant Enterococcus therapy. Strains of E. faecium Com12 displaying resistance to a particular phage encoded sagA mutations localized at the NlpC/p60 domain. Phage resistance upon sagA mutation coincided with an antibiotic fitness tradeoff that was exploited to discover the synergistic effect of phage-antibiotic combination therapy on these strains. We show that while the phage resistant strains share similar SagA secretion and cell wall profiles compared to wild type, the respective mutations abrogate NlpC/p60 hydrolase activity in vitro. These results imply that the phage resistant E. faecium Com12 strains produce catalytically impaired SagA mutants, which manifests as enhanced antibiotic susceptibility and raises the possibility of targeting NlpC/p60 hydrolase activity to treat multi-drug resistant strains of E. faecium. In Chapter 6, we identify and biochemically characterize two NlpC/p60 proteins, 501F and 51B9 CwlT, as candidate multi-drug resistant E. faecium targets. Using our established biochemical methods, we confirm that the proteins are functional peptidoglycan hydrolases in vitro, thus laying the groundwork for future functional studies of these enzymes in pathogenic E. faecium. Our results collectively indicate that NlpC/p60 hydrolase activity may serve as a marker for the viability of pathogenic bacteria. In Chapter 7, we summarize our work on NlpC/p60 hydrolases from commensal and pathogenic bacteria. We also discuss our ongoing efforts to develop high throughput activity assays for NlpC/p60 hydrolases and describe the next frontier of these enzymes as multi-drug resistant Enterococcus targets. Together, the work described in this thesis underscore the importance of NlpC/p60 peptidoglycan hydrolase activity in mediating host-microbe interactions
The Role of Compartmentalized Metabolism in Cellular Metal Homeostasis
Get PDFThe building blocks of cells are usually thought to be DNA, RNA, and proteins. However, life, as we know it, is not possible without iron. While the list of iron\u27s vital cellular functions is extensive, iron is also quite cytotoxic. Thus, great pains have been taken, at the gene regulation level, to assure that a cell has sufficient iron to copy its genome and power its mitochondria but not too much to damage its membranes with lipid peroxides. Cellular organelles, which accompanied the rise of atmospheric oxygen and an increased need for iron, also play a key role in iron homeostasis. Mitochondria are the sites of iron assimilation whereas lysosomes, with their v-ATPase-generated acidic lumens, are responsible for iron uptake and rely. In the first part of this work, I focused on lysosomes. Unbiased genetic screens performed on cells grown at sub-lethal levels of lysosomal pH inhibition identified several important metabolic pathways in this context. These included central carbon metabolism, cholesterol synthesis and iron homeostasis. While, cells starve for cholesterol and iron, only iron supplementation was necessary and sufficient to restore cell proliferation upon genetic or pharmacologic v-ATPase inhibition. Interestingly, iron supplementation rescued cell viability independent of lysosomal associated functions including signaling and endocytosis. It did, however, reverse changes resulting from low cellular iron including destabilized iron sulfur cluster proteins, induced hypoxia signaling, and impaired respiration. Finally, due to compromised aconitase activity, I identified an increased dependence on pyruvate-derived citrated as a metabolic ramification of lysosomal dysfunction. Taken together, this strongly argued that providing cellular iron is the essential function of lysosomal acidity for cell proliferation In the second part of this work, I explored other organelles in the setting of altered cellular iron. Using unbiased genetic screens, I found that iron chelation necessitates a fully intact mitochondrial iron import system. In this context, Golgi manganese uptake and storage were also essential. Because chemical or genetic induced manganese overload phenocopied iron starvation, cells were more sensitives to iron starvation and resistant to iron overload and ferroptosis. As mitochondria are the main sites of iron assimilation, I also characterized mitochondrial proteomic changes upon altered cellular iron. Here, I identified a mitochondrial solute transporter, SLC25A39, whose protein stability was proportional to cellular iron levels. Further investigation found a key role for this protein in maintaining mitochondrial GSH levels. Finally, I found that damaged or liberated iron sulfur clusters, rather than free iron, determine SLC25A39 stability. This finding may also represent an organelleautonomous regulatory loop in which mitochondria coordinate GSH and iron homeostasis
Welch\u27s Office
No full textWelch\u27s Office, n.d.
Courtesy of Medical Archives of The Johns Hopkins Medical Institutions
In one direction Welch fell further and further behind. One perquisite of every great influential had come to be possession of a scholarly journal, and Welch founded in 1896 a much needed Journal of Experimental Medicine. In a short time contributors began to find that their manuscripts had been swallowed up unto some kind of abyss and were never heard from again. Frantic letters asking for the return of the article for publication elsewhere met with no response. Hurd would sometimes scavenge through Welch’s study and retrieve a manuscript – in Welch’s absence – but the situation rapidly became hopeless. In the end, to Welch’s great relief, possession of the Journal passed from Johns Hopkins to the Rockefeller Institute in 1902. - Donald Fleming. William H. Welch and the rise of modern medicine. Boston, 1954https://digitalcommons.rockefeller.edu/jem-the-beginnings/1018/thumbnail.jp
Details of the Exhibit
No full textDetails of the exhibit JEM: The Beginnings
Idea, design - Olga Nilova, Special Collections Librarian
Photograph - Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/jem-the-beginnings/1006/thumbnail.jp
Board of Scientific Directors
No full textThe original Board of Scientific Directors of The Rockefeller Institute
Left to right: Theobald Smith, Herman M. Biggs, Simon Flexner, William H. Welch, T. Mitchell Prudden, L. Emmett Holt, Christian Herter
Courtesy of the Rockefeller Archive Center
In October 1902, Welch appealed to the board of The Rockefeller Institute to take over the Journal of Experimental Medicine. The transfer of ownership and publication responsibilities required the physical transfer of manuscripts from Welch’s office, which fell to the director of The Rockefeller Institute, Simon Flexner, who carried the abandoned manuscripts from Baltimore to New York in a suitcase.
The first issue of JEM published by The Rockefeller Institute appeared in February 1905, with Flexner serving as editor, and the journal has been published regularly since then. Although the journal was adopted by The Rockefeller Institute as a venue for publication of the Institute’s own research, it also accepted submissions from outside. Even in the early years, more than half of the papers published in the journal came from external labs.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1033/thumbnail.jp
Accessory Nucleases Provide Robust Antiparasite Immunity for Type III CRISPR-Cas Systems
Get PDFTo protect against parasites like bacteriophages and plasmids, bacteria employ diverse and sophisticated defence systems. Clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas systems are adaptive immune systems that can integrate short spacers from a parasite into its CRISPR locus as a form of immunological memory. Upon reinfection, short RNAs transcribed from the CRISPR locus can guide Cas proteins to the viral genome through complementary base pairing. Cas nucleases then destroy the invader\u27s genome. To date, six major types and multiple subtypes of CRISPR systems exist, each with their own signature genes and mechanisms of action. Type III CRISPR systems are uniquely able to destroy both the parasite\u27s DNA and RNA. Type III loci contain Cas10 and Csm2-5, which make up the main Cas10-Csm targeting complex. In addition, loci typically contain an ancillary RNase, csm6 or csx1. Upon target transcription, the Cas10-Csm complex recognises a viral transcript containing a target, which activates DNase activity of Cas10, leading to the destruction of the invader. In addition, it was recently discovered that the Palm domain of Cas10 can synthesise cyclic oligoadenylate second messengers (cA). cA can activate Csm6 by binding to the latter\u27s CARF domain. In this work, I first elucidate and illuminate the role and mechanism of action of Csm6 during anti-plasmid immunity in staphylococci. I show that Csm6 is required for efficient immunity against a weakly transcribed target but is dispensable against a welltranscribed target. Moreover, in vivo, Csm6 is a non-specific RNase, targeting both host and invader transcripts. This induces a transient growth arrest in the host cell, which is relieved upon target clearance. This growth arrest buys time for the Cas10-Csm complex to eliminate the plasmid, which is required for clearance against weakly transcribed targets. Further, I expand and characterise broader arsenal of cA-activated CARF genes that type III systems use during immunity. I identify Card1, a nuclease that can degrade both ssDNA and ssRNA in vitro. These activities required divalent cations, and were activated by cA4. In Staphylococcus aureus, Card1 induces a growth arrest upon activation, and enhance anti-phage immunity. The protection is most likely primarily through the ssDNase activity, since no RNA degradation was detected in vivo. Together with collaborators, we were also able to solve the crystal structure of apo-, cA4-, and cA6- bound Card1 structures, revealing the conformational changes allowing catalysis upon ligand binding. I also identify TM-1, a transmembrane helix-CARF gene that also causes a growth arrest in S. aureus when stimulated by cA production. The mechanism of TM-1 remains to be elucidated, but likely represents the first CRISPR protection mechanism not mediated by degrading nucleic acid. Altogether, my work both deepens and broadens our understanding of the ligandmediated immune response of type III CRISPR systems. Robust immunity is obtained by coupling specific invader destruction (Cas10 DNase activity) with non-specific host and parasite growth arrest (Csm6/Card1/TM-1). This serves as a broader paradigm of how bacteria can use different catalytic activities and different systems to resist their parasites
Mechanisms for the Evolution of Superorganismality in Ants
Get PDFAnt colonies appear to behave as superorganisms; they exhibit very high levels of within-colony cooperation, and very low levels of within-colony conflict. The evolution of such superorganismality has occurred multiple times across the animal phylogeny, and indeed, origins of multicellularity represent the same evolutionary process. Understanding the origin and elaboration of superorganismality is a major focus of research in evolutionary biology. Although much is known about the ultimate factors that permit the evolution and persistence of superorganisms, we know relatively little about how they evolve. One limiting factor to the study of superorganismality is the difficulty of conducting manipulative experiments in social insect colonies. Recent work on establishing the clonal raider ant, Ooceraea biroi, as a tractable laboratory model, has helped alleviate this difficulty. In this dissertation, I study the proximate evolution of superorganismality in ants. Using focussed mechanistic experiments in O. biroi, in combination with comparative work from other ant species, I study three major aspects of ant social behaviour that provide insight into the origin, maintenance, and elaboration of superorganismality. First, I ask how ants evolved to live in colonies, and how they evolved a reproductive division of labour. A comparative transcriptomic screen across the ant phylogeny, combined with experimental manipulations in O. biroi, finds that reproductive ants have higher insulin levels than their non-reproductive nestmates, and that this likely regulates the reproductive division of labour. Using these data, as well as studies of the idiosyncrasies of O. biroi\u27s life history, I propose a mechanism for the evolution of the first colonies. It is possible that similar mechanisms underlie the evolution of reproductive division of labour in other superorganisms, and of germ-soma separation in nascent multicellular individuals. Second, I ask how ant workers assess colony hunger to regulate their foraging behaviour. I find that workers use larval signals, but not their own nutritional states, to decide how much to forage. In contrast, they use their nutritional states, but not larval signals, to decide how much to eat, suggesting that in at least some ant species, foraging and feeding have been decoupled. This evolution of colony-level foraging regulation has occurred convergently in hymenopteran superorganisms, and is analogous to the evolution of centralised regulation of foraging behaviour in multicellular animals. Finally, I ask how an iconic collective foraging behaviour – the mass raids of army ants – evolved. I find that O. biroi, a relative of army ants, forages collectively in group raids, that these are ancestral to the mass raids of army ants, and that the transition from group to mass raiding correlates with expansion in colony size. I propose that the scaling effects of increasing colony size explain this transition. It is possible that similar principles underlie the evolution of disparate collective behaviours in other animal groups and among cells within developing animals. Together, these studies illuminate the life history of O. biroi, and suggest mechanisms for the evolution of core aspects of cooperative behaviour in ant colonies. I draw comparisons to the evolution of superorganismality in other lineages, as well as to the evolution of multicellularity. I suggest that there may be additional similarities in the proximate evolutionary trajectories of superorganismality and multicellularity
Metabolic Coordination of Stem Cell Fate Controls Tumor Initiation and Tissue Repair
Get PDFTissue stem cells balance fate decisions of self-renewal and differentiation to maintain homeostasis over the lifetime of an organism, as well as to repair tissues upon injury and wounding. Disrupting the balance between self-renewal and differentiation results in pathology: excessive self-renewal at the expense of differentiation is associated with tumor initiation, whereas failure to properly self-renew leads to stem cell exhaustion and aging. Stem cell fate is under tight regulation by the surrounding microenvironment, or niche, which includes neighboring cell types, signaling molecules, extracellular matrix, and nutrients. While the role of stromal cells and the signals they produce has been extensively studied with regards to control of stem cell fate, relatively little known is about how tissue stem cells integrate extracellular nutrient availability with fate decisions. Moreover, while intracellular metabolic pathways have been shown to regulate the balance of self-renewal and differentiation, it remains unknown whether or not endogenous metabolic pathways or nutrient availability predispose stem cells towards transformation or control their responses to tissue injury. Here, I address these questions in epidermal stem cells (EpdSCs), which maintain integrity of the skin barrier over an organism\u27s life. EpdSCs are a cell of origin for squamous cell carcinomas (SCCs), amongst the most common and threatening malignancies worldwide. Additionally, EpdSCs of the interfollicular epidermis (IFE) and hair follicle (HF) respond to injuries in the skin barrier to repair the breach. First focusing on tumor initiation, I find oncogenic EpdSCs are serine auxotrophs, whose growth and self-renewal require abundant exogenous serine. When extracellular serine is limited in vitro and in vivo, EpdSCs activate de novo serine synthesis, which in turn produces the metabolite α-ketoglutarate (αKG). αKG stimulates terminal differentiation via activation of αKG-dependent dioxygenases that remove the repressive histone modification H3K27me3, which otherwise promotes EpdSC self-renewal. Accordingly, serine starvation or enforced αKG production antagonizes SCC initiation and growth. Conversely, blocking serine synthesis or repressing αKG-driven demethylation facilitates malignant progression in vivo. Finally, I extend these findings to stem cell responses to tissue injury. In the epidermis, abrasion of the IFE activates HFSCs to repair the breach, which is associated with transient hyperproliferation, migration and activation of a plasticity program termed lineage infidelity wherein HFSCs undergo a fate switch to become IFE-SCs, fueling regeneration of a non-cognate tissue. I find that environmental serine restriction accelerates HFSC-mediated wound repair, which I link specifically to acceleration of stem cell plasticity and acquisition of IFE fate. Altogether, these findings reveal that extracellular serine is a critical determinant of EpdSC fate and provide insight into how nutrient availability is integrated with stem cell fate decisions during tumor initiation and tissue repair
Centennial Calendar and Picnic Program
No full textCentennial calendar and picnic program, 2001
The Rockefeller University, the nation’s first center for biomedical research and a leader in the international scientific community, celebrated its founding on June 14, 2001, with a gala event. Throughout the centennial year, from the fall of 2000 through the fall of 2001, the University-sponsored symposia, conferences, seminars, special exhibits, and concerts.https://digitalcommons.rockefeller.edu/objects-tell-stories/1031/thumbnail.jp