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

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline

Discerning the Ambiguous Role of Missense TTN Variants in Inherited Arrhythmogenic Syndromes

The titin gene (TTN) is associated with several diseases, including inherited arrhythmias. Most of these diagnoses are attributed to rare TTN variants encoding truncated forms, but missense variants represent a diagnostic challenge for clinical genetics. The proper interpretation of genetic data is critical for translation into the clinical setting. Notably, many TTN variants were classified before 2015, when the American College of Medical Genetics and Genomics (ACMG) published recommendations to accurately classify genetic variants. Our aim was to perform an exhaustive reanalysis of rare missense TTN variants that were classified before 2015, and that have ambiguous roles in inherited arrhythmogenic syndromes. Rare missense TTN variants classified before 2015 were updated following the ACMG recommendations and according to all the currently available data. Our cohort included 193 individuals definitively diagnosed with an inherited arrhythmogenic syndrome before 2015. Our analysis resulted in the reclassification of 36.8% of the missense variants from unknown to benign/likely benign. Of all the remaining variants, currently classified as of unknown significance, 38.3% showed a potential, but not confirmed, deleterious role. Most of these rare missense TTN variants with a suspected deleterious role were identified in patients diagnosed with hypertrophic cardiomyopathy. More than 35% of the rare missense TTN variants previously classified as ambiguous were reclassified as not deleterious, mainly because of improved population frequencies. Despite being inconclusive, almost 40% of the variants showed a potentially deleterious role in inherited arrhythmogenic syndromes. Our results highlight the importance of the periodical reclassification of rare missense TTN variants to improve genetic diagnoses and help increase the accuracy of personalized medicine

Discerning the Ambiguous Role of Missense TTN Variants in Inherited Arrhythmogenic Syndromes

The titin gene (TTN) is associated with several diseases, including inherited arrhythmias. Most of these diagnoses are attributed to rare TTN variants encoding truncated forms, but missense variants represent a diagnostic challenge for clinical genetics. The proper interpretation of genetic data is critical for translation into the clinical setting. Notably, many TTN variants were classified before 2015, when the American College of Medical Genetics and Genomics (ACMG) published recommendations to accurately classify genetic variants. Our aim was to perform an exhaustive reanalysis of rare missense TTN variants that were classified before 2015, and that have ambiguous roles in inherited arrhythmogenic syndromes. Rare missense TTN variants classified before 2015 were updated following the ACMG recommendations and according to all the currently available data. Our cohort included 193 individuals definitively diagnosed with an inherited arrhythmogenic syndrome before 2015. Our analysis resulted in the reclassification of 36.8% of the missense variants from unknown to benign/likely benign. Of all the remaining variants, currently classified as of unknown significance, 38.3% showed a potential, but not confirmed, deleterious role. Most of these rare missense TTN variants with a suspected deleterious role were identified in patients diagnosed with hypertrophic cardiomyopathy. More than 35% of the rare missense TTN variants previously classified as ambiguous were reclassified as not deleterious, mainly because of improved population frequencies. Despite being inconclusive, almost 40% of the variants showed a potentially deleterious role in inherited arrhythmogenic syndromes. Our results highlight the importance of the periodical reclassification of rare missense TTN variants to improve genetic diagnoses and help increase the accuracy of personalized medicine

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline.Research funded by Consejo Superior de Investigaciones Científicas (Spanish National Research Council) (project 202020E161, CSIC-COV19). Additional partial financial support from Project CTQ2017-85378-R (AEI/FEDER, UE) from the Spanish Ministry of Science, Innovation and Universities, is acknowledged.Publisher’s versio

How the 26S Proteasome Degrades Ubiquitinated Proteins in the Cell

The 26S proteasome is the central element of proteostasis regulation in eukaryotic cells, it is required for the degradation of protein factors in multiple cellular pathways and it plays a fundamental role in cell stability. The main aspects of proteasome mediated protein degradation have been highly (but not totally) described during three decades of intense cellular, molecular, structural and chemical biology research and tool development. Contributions accumulated within this time lapse allow researchers today to go beyond classical partial views of the pathway, and start generating almost complete views of how the proteasome acts inside the cell. These views have been recently reinforced by cryo-electron microscopy and mechanistic works that provide from landscapes of proteasomal populations distributed in distinct intracellular contexts, to detailed shots of each step of the process of degradation of a given substrate, of the factors that regulate it, and precise measurements of the speed of degradation. Here, we present an updated digest of the most recent developments that significantly contribute in our understanding of how the 26S proteasome degrades hundreds of ubiquitinated substrates in multiple intracellular environments.This work was funded by Science Dreaming SL, in the frame of a private contract.Peer reviewe