Proteasome-mediated processing of Def1: a critical step in the cellular response to DNA damage

DNA damage can pose an irreversible steric block to RNA polymerase II (RNAPII), preventing transcription. RNAPII becomes stalled at DNA lesions, blocking normal repair. As a ‘last-resort’ mechanism to clear the stalled polymerase - and repair the damage - the largest subunit of RNAPII is poly-ubiquitylated and degraded. In yeast, this process is dependent on the Def1 protein, through a previously unresolved mechanism. Using a combination of yeast genetic, biochemical and cell biological techniques this thesis reports the molecular mechanism of Def1 in this process. Upon DNA damage induced RNAPII stall, Def1 becomes ubiquitylated and partially proteolytically processed by the 26S proteasome. This creates a biologically active, shorter form of Def1, termed pr-Def1. Removal of the C-terminus of Def1, which usually promotes nuclear export, results in nuclear accumulation of the N-terminal processed fragment. Nuclear pr-Def1 binds to stalled, mono-ubiquitylated RNAPII and recruits the Elongin-Cullin ubiquitin ligase complex, promoting RNAPII polyubiquitylation and degradation. Interestingly, Def1’s ubiquitin-binding CUE domain and a novel ubiquitin homology domain in the Elongin complex mediate this interaction. These results outline the multi-step mechanism of RNAPII poly-ubiquitylation, elucidate Def1 activation and function, and identify an atypical ubiquitin-like domain in the yeast Elongin complex

Chemical and biochemical studies of ubiquitin conjugation machinery

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.The post-translational modification of proteins is a major mechanism employed in eukaryotic cells to expand the functional diversity of the proteome. Covalent modification of amino acid side chains confers new or altered functionality to the modified protein by creating new recognition surfaces on the protein for the interaction of nucleic acids or other proteins, modulating enzymatic activity, or altering cellular localization or half-life. The post-translational modification of proteins with ubiquitin (Ub) is an important mechanism of regulating protein function. Ub is a 76-residue protein that is primarily attached to lysine residues in target proteins through an enzymatic cascade catalyzed by E1, E2, and E3 enzymes. Ub conjugation is important for fundamental cellular processes, including transcription, DNA repair, endocytosis, apoptosis, and signal transduction. Ub conjugation is reversible. Proteases termed deubiquitinating enzymes (DUBs) function to remove Ub from target proteins. Genome sequencing efforts have uncovered the existence of many predicted enzymes with unknown function. Many enzymes have been assigned function based on sequence homology to proteins with known function without confirmation of enzymatic activity. A powerful chemical approach to determine enzyme function from a complex mixture of proteins is activity-based protein profiling. This method makes use of chemical probes that are active site-directed for the assignment of function to proteins. We describe here the design and generation of an expanded set of Ub-based chemical probes with which we identified and recovered E1, E2, and E3 Ub ligases from cell lysates. Furthermore, we describe the biochemical and structural characterization of the catalytic domain of one E3 Ub ligase we recovered, HUWE1, and the identification of a structural element within the catalytic domain of HUWE1 that modulates its activity. Finally, we discuss a protein engineering method that we are applying to the HUWE1 catalytic domain to understand how the conformational flexibility of this domain is important to its function.by Renuka K. Pandya.Ph.D

Trafficking and Quality Control of Inward-Rectifying Potassium Channels in the Model Eukaryote Saccharomyces cerevisiae

Humans express 15 different inward-rectifying potassium (Kir) channels that play a variety of roles in epithelial transport, nerve conduction, and muscle contraction. Polymorphisms in Kir channels underlie numerous genetic diseases. In order to investigate the function and regulation of Kir channels, researchers have long recognized the potential of expressing them in strains of Saccharomyces cerevisiae (Bakers’ yeast) engineered for sensitivity to growth in a low potassium medium. These studies capitalize on the wide variety of genetic and biochemical tools available in yeast, namely the ease of transforming large quantities of cells with recombinant DNA and the availability of comprehensive mutant libraries for high-throughput screening. Due to the longstanding popularity of yeast as model eukaryotes, the mechanisms underlying cellular phenomena are relatively well-documented and are also conserved in multicellular organisms. In this document, I will first review the history and diversity of techniques for studying potassium channels in potassium-sensitized yeast. Next, I will describe the results of a genetic screen to identify the key regulatory factors for a particular Kir channel: the renal outer medullary potassium (ROMK) channel. ROMK, also known as Kir1.1, is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. I used a synthetic genetic array to identify nonessential genes that reduce the plasma membrane pool of ROMK in potassium uptake-deficient yeast. Through this screen, I identified several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in a mammalian cell line, therefore establishing that post-endocytic sorting influences the cell-surface density of ROMK and modulates its activity. Finally, I will briefly delineate potential future studies in yeast with ROMK and other Kir channels and will describe several investigations into various aspects of Kir channel biology to which I have contributed through collaboration

Μελέτη των ρυθμιστικών μηχανισμών στόχευσης και ενδοκύτωσης διαμεμβρανικών μεταφορέων πουρινών σε ένα πρότυπο γενετικό σύστημα

Οι ευκαρυωτικοί διαμεμβρανικοί μεταφορείς ανταποκρίνονται σε περιβαλλοντικά και αναπτυξιακά σινιάλα τόσο στο μεταγραφικό όσο και στο μετα-μεταφραστικό επίπεδο. Η έκφραση των μεταφορέων ρυθμίζεται αυστηρά με την ταχεία de novo σύνθεση και στόχευση τους στη πλασματική μεμβράνη, αλλά και την ακόμη πιοταχεία απομάκρυνσή τους απο αυτήν μέσω της διαδικασίας της ενδοκύτωσης, ως απόκριση στην παρουσία ιόντων αμμωνίου ή περίσσειας υποστρώματος. Ο εκτενώς μελετημένος μεταφορέας ουρικού οξέος-ξανθίνης UapA του Aspergillus nidulans χρησιμοποιήθηκε για τη διερεύνηση τριών βασικών ερωτημάτων που σχετίζονται με τους μηχανισμούς που διέπουν την ενδοκυτταρική διακίνηση των μεταφορέων. Συγκεκριμένα, μελετήθηκαν οι συνέπειες του υπερτονικού στρες στην φυσιολογία των μυκήτων και την ενδοκύτωση των μεταφορέων τους, οι μηχανισμοί που ρυθμίζουν την ουβικουιτινίωση των μεταφορέων με σκοπό την απομάκρυνση τους από την πλασματική μεμβράνη και την καταστροφή τους στα χυμοτόπια, και ο ρόλος του ολιγομερισμού στην διακίνηση των μεταφορέων προς και από την πλασματική μεμβράνη.Eukaryotic transporters respond to environmental and developmental signals at both the transcriptional and post-translational levels. Their tight control includes rapid de novo synthesis and targeting to the plasma membrane (PM) and even more rapid down-regulation through endocytosis and vacuolar degradation, triggered by NH4 + orexcess substrate. The extensively studied uric acid-xanthine transporter UapA of Aspergillus nidulans was used to address three questions concerning the mechanisms underlying intracellular trafficking of transport proteins: what are the effects of hypertonicity in fungal physiology and transporter endocytosis, which are the mechanisms regulating transporter ubiquitination and turnover, and what is the role of transporter oligomerization in membrane trafficking and endocytosis

Μελέτη των ρυθμιστικών μηχανισμών στόχευσης και ενδοκύτωσης διαμεμβρανικών μεταφορέων πουρινών σε ενα πρότυπο γενετικό σύστημα

Οι ευκαρυωτικοί διαμεμβρανικοί μεταφορείς ανταποκρίνονται σε περιβαλλοντικά και αναπτυξιακά σινιάλα τόσο στο μεταγραφικό όσο και στο μετα-μεταφραστικό επίπεδο. Η έκφραση των μεταφορέων ρυθμίζεται αυστηρά με την ταχεία de novo σύνθεση και στόχευση τους στη πλασματική μεμβράνη, αλλά και την ακόμη πιο ταχεία απομάκρυνσή τους απο αυτήν μέσω της διαδικασίας της ενδοκύτωσης, ως απόκριση στην παρουσία ιόντων αμμωνίου ή περίσσειας υποστρώματος. Ο εκτενώς μελετημένος μεταφορέας ουρικού οξέος-ξανθίνης UapA του Aspergillus nidulans χρησιμοποιήθηκε για τη διερεύνηση τριών βασικών ερωτημάτων που σχετίζονται με τους μηχανισμούς που διέπουν την ενδοκυτταρική διακίνηση των μεταφορέων. Συγκεκριμένα, μελετήθηκαν οι συνέπειες του υπερτονικού στρες στην φυσιολογία των μυκήτων και την ενδοκύτωση των μεταφορέων τους, οι μηχανισμοί που ρυθμίζουν την ουβικουιτινίωση των μεταφορέων με σκοπό την απομάκρυνση τους από την πλασματική μεμβράνη και την καταστροφή τους στα χυμοτόπια, και ο ρόλος του ολιγομερισμού στην διακίνηση των μεταφορέων προς και από την πλασματική μεμβράνη

Identification and Functional Studies of Arabidopsis thaliana Ubc13-interacting E3 Ubiquitin Ligases

In eukaryotic organisms, polyubiquitination is the modification of a protein with polymerized ubiquitin (Ub) chain. This process is well known for its function in targeting proteins for degradation by the 26S proteasome. However, a polyUb chain assembled through the lysine 63 residue of the Ub moiety (Lys63-linked polyubiquitination) has been shown to play a signaling role rather than targeting proteins for degradation. In plants, the functions of Lys63-linked polyubiquitination are currently not well understood. Ub-protein ligase (E3) catalyzes the last step in the ubiquitination reactions, and to a large extent it also determines the substrate specificity of protein ubiquitination. In order to study the roles of Lys63-linked polyubiquitination in plants, two E3s of Arabidopsis thaliana, proteins encoded by AtCHIP and At1g74370 (tentatively named E3-A1), were chosen for functional studies, since they interacted with AtUbc13A protein. Sequence analysis showed that AtCHIP is the only member in the family. A T-DNA insertion mutant line (Atchip-1) was obtained to study the AtCHIP gene knock-out effect. The mutant line was grown in normal conditions and further tested in a variety of conditions: hormonal treatments, osmotic stress, seed deterioration, high temperature stress, high-intensity light stress, oxidative stress and DNA damaging stress. However, no clear difference was observed between the mutant and wild type plants based on the several parameters measured. Sequence analysis of E3-A1 indicated two closely related proteins, tentatively named E3-A2 and E3-A3. As E3-A1 and E3-A2 appeared to share more sequence similarity, RNA interference (RNAi) transformants, with the level of transcripts for either of the two E3-A genes reduced by over 90% were generated. Selected RNAi mutant lines for E3-A1 and E3-A2 were crossed with each other, and double RNAi mutants were obtained. However, no distinct phenotype was detected under normal, high-sucrose or hormonal conditions for either single or double RNAi lines. Although various assays did not reveal a significant phenotype in the mutants in this study, the materials generated and the assays used will benefit a wider range of phenotypic survey in the future

Structure/function study of the proteasome: characterization of Rpn5 sumoylation and development of proteolytic chimeras

El sistema ubicuitina-proteasoma és una via reguladora central en l'homeòstasi de proteïnes. Funciona específicament etiquetant proteïnes amb ubicuitina i enviant-les al proteasoma per a la seva degradació. Aleshores, el proteasoma reconeix l'etiqueta mitjançant receptors d'ubicuitina, desplega la proteïna diana i la degrada tallant-la en petits pèptids. Dos sub-complexos proteasòmics principals controlen el procés de degradació de proteïnes: el centre proteolític del proteasoma està contingut en una estructura proteica cilindrica en forma cilíndrica anomenada partícula nucli (PC). Per altra banda, la partícula reguladora (PR) conté receptors d'ubicuitina, enzims deubicuitinitzants i ATPases que fan la força de tracció necessària per desplegar els substrats. La forma en què el proteasoma captura, desplega i degrada la proteïna requereix que el complex experimenti una sèrie de canvis d'estat conformacional amb l'objectiu de passar d'un estat de repòs, en el qual la majoria dels centres catalítics romanen inactius o no compromesos, a un estat totalment actiu on les proteïnes són fàcilment processades i degradades. En el context del proteasoma, Rpn5 (PSMD12 en humans) és una subunitat integrant de la tapa de la partícula reguladora, funcionant com a bastida per al muntatge d'aquest subcomplex, i en proteasomes madurs es situa en contacte amb la base i la tapa per transmetre els canvis de moviment que permeten el funcionament del proteasoma. En aquest projecte hem caracteritzat la SUMOilació de Rpn5 en llevats, un esdeveniment descrit inicialment en PSMD12 humà, per tal d'entendre les seves implicacions biològiques i relacions estructura/funció. Hem determinat que Rpn5 està preferentment SUMOilat a la lisina 147. Utilitzant una fusió Smt3-Rpn5 com a model per a aquesta modificació posterior a la traducció, hem detectat que els proteasomes SUMOilats adopten una configuració inusual, amb quantitats disminuïdes de partícula central. Sorprenentment, però, es mantenen les capacitats de degradació del substrat dels proteasomes, cosa que suggereix un augment compensatiu de l'estat d'activació entre els proteasomes SUMOilats. Quan s’han analitzat les implicacions biològiques d'aquesta troballa, vam trobar diferències en les proteïnes que interaccionen amb el proteasoma, cosa que indica un canvi de preferència de substrat pels proteasomes SUMOilats. A més, en assajos fenotípics, la sumoilació del proteasoma es va relacionar amb una sensibilitat al zenc. Al mateix temps, a les soques amb deficiència de l’enzim deubicuitinitzant Ubp6, la SUMOilació Rpn5 va rescatar fenotips intrínsecs, cosa que indica una interacció entre la SUMOilació Rpn5 i la regulació dels enzims deubicuitinitzants associats al proteasoma. És important destacar que vam obtenir una estructura crio-EM amb 8Å de resolució de proteasomes SUMOilats, que va revelar que aquests proteasomes modificats amb SUMO estaven preferentment en estat activat. Paral·lelament a la caracterització de la SUMOilació d’Rpn5, aquest projecte va explorar la viabilitat d'utilitzar components del proteasoma com a receptors per a degradadors quimèrics de tipus protac. Els protacs tradicionals consisteixen en molècules bifuncionals unides per un enllaç, amb l'objectiu d'aproximar una ubicuitina lligasa (E3) i una proteïna a degradar. Després de la formació d'un complex ternari, la proteïna objectiu és ubicuitilada i després reconeguda i degradada pel proteasoma. En un experiment de prova de concepte, vam desenvolupar el compost RBM3-300, una molècula capaç de guiar la inosina monofosfat deshidrogenasa 2 (IMPDH2) a la seva degradació pel proteasoma. Després de 72 h de tractament amb el compost a una concentració de 10 µM, vam poder determinar una reducció dels nivells d'IMPDH2 en cultius de mamífer. A més, vam determinar les constants d'afinitat d'aquest compost principal, amb altres que contenen diferents característiques químiques, cap als seus receptors de proteïnes mitjançant ressonància plasmònica de superfície. Finalment, com a segona prova de concepte, vam fer un assaig de degradació amb proteasomes que tenien una proteïna de fusió sensible a la glutamina i gliadina de blat com a model d'un substrat ric en glutamina. Vam trobar que la combinació d'això amb l'ablació mitjançada per CRISPR dels extrems N-terminals de la subunitat alfa que tanquen la partícula central, dona lloc a proteasomes que han vist augmentada la processivitat d'aquests substrats.El sistema ubiquitina-proteasoma es una vía reguladora central en la homeostasis de proteínas. Funciona específicamente etiquetando proteínas con ubiquitina y enviándolas al proteasoma para su degradación. Entonces, el proteasoma reconoce la etiqueta mediante receptores de ubiquitina, despliega la proteína diana y la degrada cortándola en pequeños péptidos. Dos sub-complejos proteasómicos principales controlan el proceso de degradación de proteínas: el centro proteolítico del proteasoma está contenido en una estructura proteica cilíndrica con forma cilindrica denominada partícula núcleo (PN). Por otro lado, la partícula reguladora (PR) contiene receptores de ubiquitina, enzimas deubiquitinizantes y ATPasas que realizan la fuerza de tracción necesaria para desplegar los sustratos. La forma en que el proteasoma captura, despliega y degrada la proteína requiere que el complejo experimente una serie de cambios de estado conformacional con el objetivo de pasar de un estado de reposo, en el que la mayoría de los centros catalíticos permanecen inactivos o no comprometidos, a un estado totalmente activo en el que las proteínas son fácilmente procesadas y degradadas. En el contexto del proteasoma, Rpn5 (PSMD12 en humanos) es una subunidad integrante de la tapa de la partícula reguladora, funcionando como andamio para el montaje de este subcomplejo, y en proteasomas maduros se sitúa en contacto con la base y la tapa para transmitir los cambios de movimiento que permiten el funcionamiento del proteasoma. En este proyecto hemos caracterizado la SUMOilación de Rpn5 en levaduras, un evento descrito inicialmente en PSMD12 humano, para entender sus implicaciones biológicas y sus relaciones estructura/función. Hemos determinado que Rpn5 está preferentemente SUMOilado en la lisina 147. Utilizando una fusión Smt3-Rpn5 como modelo para esta modificación posterior a la traducción, detectamos que los proteasomas SUMOilados adoptan una configuración inusual, con cantidades disminuidas de partícula central. Sorprendentemente, sin embargo, se mantienen las capacidades de degradación del sustrato de los proteasomas, lo que sugiere un aumento compensativo del estado de activación entre los proteasomas SUMOilados. Cuando se analizaron las implicaciones biológicas de este hallazgo, encontramos diferencias en las proteínas que interaccionan con el proteasoma, indicando un cambio de preferencia de sustrato por los proteasomas SUMOilados. Además, en ensayos fenotípicos, la SUMOilación del proteasoma se relacionó con una sensibilidad en el Zn. Al mismo tiempo, en las cepas con deficiencia de deubiquitilasa UBP6, la sumoilación Rpn5 rescató fenotipos intrínsecos, lo que indica una interacción entre la SUMOilación Rpn5 y la regulación de las enzimas deubiquitilantes asociadas al proteasoma. Es importante destacar que obtuvimos una estructura crio-EM con 8Å de resolución de proteasomas SUMOilados, que reveló que estos proteasomas modificados con SUMO estaban preferentemente en estado activado. Paralelamente a la caracterización de la SUMOilación de Rpn5, este proyecto exploró la viabilidad de utilizar componentes del proteasoma como receptores para degradadores quiméricos de tipo protac. Los protacs tradicionales consisten en moléculas bifuncionales unidas por un enlace, con el objetivo de aproximar una ubiquitina ligasa (E3) y una proteína a degradar. Tras la formación de un complejo ternario, la proteína objetivo es ubiquitilada y después reconocida y degradada por el proteasoma. En un experimento de prueba de concepto, desarrollamos el compuesto RBM3-300, una molécula capaz de guiar la inosina monofosfato deshidrogenasa 2 (IMPDH2) hacia su degradación por el proteasoma. Después de 72 h de tratamiento con el compuesto a una concentración de 10 µM, pudimos determinar una reducción de los niveles de IMPDH2 en cultivos de mamífero. Además, determinamos las constantes de afinidad de este compuesto principal, con otras que contienen distintas características químicas, hacia sus receptores de proteínas mediante resonancia plasmónica de superficie. Por último, como segunda prueba de concepto, realizamos un ensayo de degradación con proteasomas que tenían una proteína de fusión sensible a la glutamina y gliadina de trigo como modelo de un sustrato rico en glutamina. Encontramos que la combinación de ello con la ablación intermediada por CRISPR de los extremos N-terminales de la subunidad alfa que cierran la partícula núcleo, da lugar a proteasomas que han visto aumentada la procesividad de estos sustratos.The Ubiquitin-Proteasome System is a central regulatory pathway for protein homeostasis. It functions specifically tagging proteins with ubiquitin and sending them to the proteasome for their degradation. The proteasome then recognizes the ubiquitin tag by means of receptors, unfolds the target protein and degrades it by cutting it into small peptides. Two main proteasomal subassemblies control protein degradation process: the core particle, the proteolytic centre of the proteasome enclosed in the barrel-shaped cylinder, and the regulatory particle, which contains ubiquitin receptors, deubiquitinating factors and ATPases that do the pull force to unfold substrates. The way in which the proteasome engages, unfolds and degrades protein requires the complex to undergo a series of conformational state shifts with the objective of shifting from a resting state, in which most of catalytic centres remain inactive or unengaged, to a fully active state, un which proteins are readily processed and degraded. In the context of the proteasome, Rpn5 (PSMD12 in humans) is placed in the lid of the regulatory particle, working as a scaffolding for the assembly of this subcomplex, and in mature proteasomes it contacts base and lid to transmit the motion changes that enable the functioning of the proteasome. In this project we have characterized Rpn5 sumoylation in yeast, an event initially described in human PSMD12, in order to understand its biological and mechanistical implications. We have determined that Rpn5 is preferentially SUMOylated in Lysine 147. Using a Rpn5-Smt3 fusion as a model for this post translational modification, we detected that SUMOylated proteasomes adopt an unusual configuration, with decreased amounts of core particle. Surprisingly, however, the substrate degrading capabilities of the proteasomes were maintained, suggestive of a compensative increased state of activation among SUMOylated proteasomes. When looking at the biological implications of this finding, we found differences in the proteasome interacting proteins, indicating a change of substrate preference by SUMOylated proteasomes. Additionally, in phenotypic assays, proteasome sumoylation was related to a Zn sensitivity. At the same time, in UBP6 deubiquitylase-defficient strains, Rpn5 sumoylation rescued intrinsic phenotypes, indicating an interplay between Rpn5 SUMOylation and the regulation of proteasome-associated deubiquitylating enzymes. Importantly, we obtained an 8Å resolution cryo-EM structure of SUMOylated proteasomes, which revealed that these SUMO modified proteasomes were preferentially in an activated state. In parallel to the characterization of Rpn5 SUMOylation, this project explored the feasibility of using proteasome components as receptors for protac-like chimeric degraders. Traditional protacs consist in bifunctional molecules joined by a linker, with the objective of bringing together an E3 ubiquitin ligase and a protein to be degraded. Upon formation of a ternary complex, the target protein is ubiquitylated and then recognised and degraded by the proteasome. In a proof-of-concept experiment, we developed lead compound RBM3-300, a molecule capable of targeting inosine monophosphate dehydrogenase 2 (IMPDH2) to its degradation by the proteasome. After 72h of treatment with 10µM compound, we could determine a reduction of IMPDH2 levels in mammalian cultures. Additionally, we determined the affinity constants of this lead compound, with others containing different chemistries, towards their protein receptors using surface plasmon resonance. Finally, as a second proof of concept, we challenged proteasomes bearing a glutamine-sensing fusion with the degradation wheat gliadin as model of a glutamine rich substrate. We found that combining this with CRISPR mediated ablation of the alpha subunit N-terminal ends that gate the Core Particle, results in engineered proteasomes that have increased processivity of these substrates