Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway.

In eukaryotes, the ubiquitin-proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore-1 loops of the RP's Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N-terminus, remain unknown. We determined how the location of glycine-rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine-rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate.

Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results imply that phase separation can act as a regulatory switch that controls the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code.

The importance of proteasome grip depends on substrate stability.

The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto substrates and unfolds them by pulling them through a central pore and translocating them into the 20S degradation chamber. A set of pore loops containing a so-called aromatic paddle motif in each Rpt subunit is believed to be important for the proteasome's ability to unfold and translocate substrates. Based on structural and mechanistic experiments, paddles from adjacent Rpt subunits, which are arrayed in a spiral staircase conformation, grip and pull on the substrate in a hand-over-hand type mechanism, disengaging at the bottom of the staircase and re-engaging at the top. We tested the contribution of the aromatic paddles to unfolding substrates of differing stabilities by mutating the paddles singly or in combination. For an easy-to-unfold substrate (a circular permutant of green fluorescent protein; GFP), mutations had little effect on degradation rates. For a substrate with moderate stability (enhanced GFP), there were modest effects of individual mutations on GFP unfolding rates, and alternating aromatic paddle mutants had a larger detrimental effect on unfolding than sequential mutants. For a more stable substrate (superfolder GFP), unfolding is overall slower, and multiple simultaneous mutations essentially prevent unfolding. Our results highlight the context-dependent need for grip during unfolding, support the hand-over-hand model for substrate unfolding and translocation, and suggest that for hard-to-unfold substrates, it is important to have simultaneous strong contacts to the substrate for unfolding to occur. The results also suggest a kinetic proofreading model, where substrates that cannot be easily unfolded are instead clipped, removing the initiation region and preventing futile unfolding attempts.

The 26S Proteasome Switches between ATP-Dependent and -Independent Mechanisms in Response to Substrate Ubiquitination.

The ubiquitin-proteasome system is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome through the attachment of polyubiquitin chains. Several proteins also contain ubiquitin-independent degrons (UbIDs) that allow for proteasomal targeting without the need for ubiquitination. Our laboratory previously showed that UbID substrates are less processively degraded than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust, with complete degradation only occurring with loosely folded substrates. UbID degradation was unaffected by the nonhydrolyzable ATP analog ATPγS, indicating that UbID degradation proceeds in an ATP-independent manner. Stabilizing substrates halted UbID degradation, indicating that the proteasome can only capture UbID substrates if they are already at least transiently unfolded, as confirmed using native-state proteolysis. The 26S proteasome therefore switches between ATP-independent weak degradation and ATP-dependent robust unfolding and degradation depending on whether or not the substrate is ubiquitinated.

Determination of Proteasomal Unfolding Ability.

We use an in vitro degradation assay with a model substrate to assess proteasomal unfolding ability. Our substrate has an unstructured region that is the site of ubiquitination, followed by an easy-to-unfold domain and a difficult-to-unfold domain. Degradation proceeds through the unstructured and easy-to-unfold domains, but the difficult-to-unfold domain can be degraded completely or, if the proteasome stalls, can be released as a partially degraded fragment. The ratio between these two possible outcomes allows us to quantify the unfolding ability and determine how processively the proteasome degrades its substrates.

Proteasomal conformation controls unfolding ability.

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome's various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome's ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome's conformational state and its unfolding ability.

An Integrated Computational and Experimental Approach to Identifying Inhibitors for SARS-CoV-2 3CL Protease.

The newly evolved SARS-CoV-2 has caused the COVID-19 pandemic, and the SARS-CoV-2 main protease 3CLpro is essential for the rapid replication of the virus. Inhibiting this protease may open an alternative avenue toward therapeutic intervention. In this work, a computational docking approach was developed to identify potential small-molecule inhibitors for SARS-CoV-2 3CLpro. Totally 288 potential hits were identified from a half-million bioactive chemicals via a protein-ligand docking protocol. To further evaluate the docking results, a quantitative structure activity relationship (QSAR) model of 3CLpro inhibitors was developed based on existing small molecule inhibitors of the 3CLproSARS- CoV- 1 and their corresponding IC50 data. The QSAR model assesses the physicochemical properties of identified compounds and estimates their inhibitory effects on 3CLproSARS- CoV- 2. Seventy-one potential inhibitors of 3CLpro were selected through these computational approaches and further evaluated via an enzyme activity assay. The results show that two chemicals, i.e., 5-((1-([1,1'-biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione and N-(4-((3-(4-chlorophenylsulfonamido)quinoxalin-2-yl)amino)phenyl)acetamide, effectively inhibited 3CLpro SARS-CoV-2 with IC50's of 19 ± 3 µM and 38 ± 3 µM, respectively. The compounds contain two basic structures, pyrimidinetrione and quinoxaline, which were newly found in 3CLpro inhibitor structures and are of high interest for lead optimization. The findings from this work, such as 3CLpro inhibitor candidates and the QSAR model, will be helpful to accelerate the discovery of inhibitors for related coronaviruses that may carry proteases with similar structures to SARS-CoV-2 3CLpro.

Mode of targeting to the proteasome determines GFP fate.

The ubiquitin-proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.

Ubiquitin receptors are required for substrate-mediated activation of the proteasome's unfolding ability.

The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome's unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome's unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

En torno a la supervisión oficial / Around the official supervision / Au tours de la supervision officielle

Junto con el análisis personal y los seminarios, la supervisión es uno de los pilares en los que se sostiene la formación del analista. Como en su escucha el analista se enfrenta con problemas tanto epistemológicos como epistemofílicos, la supervisión se convierte en el espacio para entender no solo el modo en que se articula la clínica con la teoría sino de entender el manejo de los fenómenos psicoanalíticos que involucran a un analista con su paciente; una clínica que se produce bajo transferencia. Entiendo que mi tarea como supervisor no se debe centrar en la interpretación del discurso del paciente a través del relato que trae el supervisando, sino en ayudarlo a pensar el propio relato con el que presenta a su paciente tanto en lo que se refiere a sus interpretaciones como a aquellos puntos ciegos a los que se enfrenta con su escucha

Together with personal analysis and seminars, supervision is one of the pillars underpinning the analyst's training. Just as in analytic listening the analyst is confronted with problems both epistemological and epistemophilic, supervision becomes a space for understanding not only the manner in which clinical practice articulates with theory, but for understanding how psychoanalytic phenomena operate, involving an analyst with his/her patient; a psychopathology that emerges under transference. In my understanding, my task as supervisor should not focus on the interpretation of the patient's discourse via the account provided by the supervisee, rather on helping the supervisee to think about the actual account s/he presents the patient with, both in terms of interpretations as well as those blind points si he comes up against in the act of listening

Avec l'analyse personnelle et les séminaires, la supervision est un des piliers qui soutient la formation de l' analyste. Comme dans son écoute l'analyste est confronté a des problemes autant épistémologiques qu'épistémophiliques, la supervision devient l'espace pour comprendre non seulement la façon dont s' articule la clinique avec la théorie mais également pour comprendre la gestion des phénomenes psychanalytiques qu'impliquent un analyste avec son patient; une clinique qui se produit sous transfert. Je comprends que mon travail comme superviseur ne doit pas se centrer dans l'interprétation du discours du patient a travers le récit qu'apporte le supervisant, mais de l'aider a penser le récit qu'il présente de son patient autant en ce qui concerne ses interprétations comme ses taches aveugles auxquelles il fait face avec son écoute

Lo íntimo y lo éxtimo en el sujeto del inconsciente / The intimate and the extimate in the subject of the unconscious / L'intime et l'extime dans le sujet de l'inconscient

Se recorre el concepto de extimidad creado por J. Lacan en consonancia con su teorización de que el inconsciente es el discurso del otro. Es una noción paradojal que implica que lo más íntimo proviene de algo ajeno a él. En la clínica se constata en el síntoma, la transferencia y en las formaciones del inconsciente en general. Freud ya comenzó a vislumbrar a lo más íntimo como lo más ajeno, tanto en La interpretación de los sueños como en El proyecto. Lo éxtimo designa un centro exterior en el yo que ninguna identificación imaginaria o simbólica puede remedar porque se trata de una falta constituyente en relación con la palabra. Así lo íntimo y lo éxtimo constituyen una unidad estructural necesaria que da cuenta de la división subjetiva, que impide hablar de una mismidad de un uno sin el otro, y que por tanto subvierte el concepto del sujeto como individuo

The article summarizes Lacan's concept of extimacy (extimité), consonant with his theory that "the unconscious is the discourse of the other". This paradoxical notion suggests that that which is most intimate derives from something outside of oneself. In a clinical setting, this may show itself in the symptom, transference and more generally in the formations of the unconscious. Freud had already begun to see the most intimate as the most alien, both in the interpretation of dreams and in his Project. The extimate designates an external centre in the ego that no imaginary or symbolic identification can emulate because it relates to a constitutive fault in relation to the word. The intimate and the extimate as such constitute a necessary structural unit which accounts for subjective division, making it difficult to speak of a selfhood of one without the other, and which therefore subverts the concept of the subject as an individual

On parcourt le concept d' extimité créé par J. Lacan en consonance avec sa théorisation de l'inconscient comme discours del' autre. C' est une notion paradoxale qui implique que le plus intime provient de quelque chose étranger a lui. Dans la clinique on le constate dans le symptome transfert, et dans les formations de l'inconscient en général. Freud avait déja commencé a entrevoir le plus intime comme le plus lointain, autant dans L'interprétation des rêves que dans Le projet. L' extime désigne un centre extérieur dans le moi qu'aucune indentification imaginaire ou symbolique ne peut remédier parce qu'il s'agit d'un manque constitutionnel en relation avec le mot. Ainsi l'intime et l'extime constituent une unité structurelle nécessaire qui rend compte de la division subjective, qui empeche de parler du «soi-meme» de l'un sans l'autre, et qui done subvertit le concept du sujet en tant qu' individu

Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome.

In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We find that the architecture of polyubiquitin chains attached to a substrate affects the ability of the proteasome to unfold and degrade the substrate, with K48- or mixed-linkage chains leading to greater processivity than K63-linked chains. Ubiquitin-independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin-dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. Our work supports a model in which a polyubiquitin chain associated with a substrate switches the proteasome into an activated state that persists throughout the degradation process.

Em torno do prazer e do gozo / About pleasure and jouissance / En torno del placer y del goce

Neste trabalho, bordejaremos o buraco do real. E o faremos em torno dos conceitos de prazer e de gozo. Como Freud disse, só se aprende nos casos em que a defesa fracassa, porque então há retornos do recalcado e a moção pulsional encontra um modo substitutivo para se descarregar, ainda que seja um substituto mutilado, inibido ou deslocado, que já não é reconhecível como satisfação, que não produz sensação de prazer, mas gozo. Prazer e gozo. Prazer freudiano e gozo lacaniano. Em Além do princípio do prazer, Freud diz que a parte essencial do recalcado não pode ser lembrada e que não se repete a serviço da recuperação de uma vivência prazerosa, mas a serviço de uma vivência que não pôde ser. Para Lacan, em contrapartida, o sexual é o que fica fora do discurso, do lado do gozo, que, enquanto experiência do real, fica fora da palavra. Trata-se, portanto, do gozo do corpo enquanto real, do corpo que fica fora do discurso e do qual só podemos falar através de seus semblantes. Esse gozo se produz quando o sujeito é capturado pela linguagem. É o que fica como um resto que alíngua não pode significar.

In this paper, we discuss the hole of the real, taking into consideration the concepts of pleasure and jouissance (joy). As Freud said, we only learn when our defense fails, because, in this way, the repressed returns and the instinctual impulse finds a substitute mode to relieve itself, even if it is a mutilated, inhibited, and displaced substitute, which is no longer able to be recognized as satisfaction, and does not bring pleasure but jouissance (joy). Pleasure and jouissance. Freudian pleasure and Lacanian jouissance. In his work Beyond the pleasure principle, Freud says the essential part of the repressed cannot be remembered, and it does not repeat itself in order to bring back a pleasurable experience, but it does in order to live an experience that could not be lived. On the other hand, Lacan believes it is the sexual that is excluded from the discourse - in the side of jouissance, which remains outside the word, as an experience of the real. Therefore, it is about the jouissance of the body, as a real experience, the body that remains outside the discourse, and about which we can only speak through its looks (facial expressions). This jouissance is brought forth when the subject is captured by language. This is a remainder that lalangue cannot mean.

En este trabajo bordearemos el orificio de lo real. Y lo haremos en torno a los conceptos de placer y de goce. Como dice Freud, solo es posible aprender en casos en los que la defensa fracasa porque entonces hay retornos de lo reprimido y la moción pulsional encuentra un modo sustituto para descargar, aunque se trate de un sustituto mutilado, inhibido o desplazado, que ya no se reconoce como satisfacción, que no produce una sensación de placer sino de goce. Placer y goce. Placer freudiano y goce lacaniano. Si Freud, en Más allá del principio del placer dice que lo esencial de lo reprimido no se recuerda y que no se repite al servicio de la recuperación de una vivencia placentera sino de una vivencia que no pudo ser, para Lacan, en cambio, lo sexual es lo que queda fuera del discurso, del lado del goce, que, si bien la experiencia de lo real, queda por fuera de la palabra. Se trata por lo tanto del goce del cuerpo real, del cuerpo que queda fuera del discurso y del que solo podemos hablar a través de sus semblantes. Este goce se produce cuando el sujeto es atrapado por el lenguaje. Es lo que queda como un resto que lalengua no puede significar.

Slipping up: partial substrate degradation by ATP-dependent proteases.

ATP-dependent proteases are present in all organisms, where they are responsible for much of intracellular protein degradation. Most proteins are processively unfolded and degraded into small peptides; however, in a few so-called slippery substrates, the protease stalls at a folded domain and releases a large protein fragment. In this review, we describe the properties of physiological slippery substrates that are processed in this manner by ATP-dependent proteases and the recent advances that have been made in understanding the mechanism underlying their partial degradation.

¿Qué se recuerda? ¿Qué se repite? / What is remembered, what is repeated?

Los recuerdos encubridores (todo recuerdo es encubridor) se ocupan de contrabalancear en su totalidad la amnesia infantil. La cura ya no está al servicio de producir un reencuentro entre el recuerdo y el hecho que fue, sino el de hacer surgir al recuerdo como significante para que sirva de enlace entre lo que ocurrió, lo que se vivenció, lo que se deseó y lo que el sujeto está viviendo en el presente transferencia!. Es decir, que el sujeto trae un relato de un recuerdo que, por un lado, no tiene que ver con la verdad de lo acontecido, sino con su compromiso subjetivo con el hecho. Y por otro lado, se trata de un recuerdo cuya significación solo adviene en el curso del análisis, en la experiencia analítica. Es el momento en que aparece la repetición que ya no puede ser reducida al relato del sujeto como un intento de simbolizar imaginariamente lo real y que se expresa como el dolor de ser en la transferencia, en la relación del analista en tanto otro (AU)

Screen memories (all memories are screen memories) are tasked with wholly counterbalancing infantile amnesia. The cure is no longer in the service of bringing about a reencounter between the memory and the event that was, rather of causing the memory to emerge as a signifier so that it serves as a link between what occurred, what was experienced, what was desired and what the subject is experiencing in the transferential present. In other words, the subject brings an account of a memory that on the one hand has nothing to do with the truth of what took place, rather his or her subjective involvement in the event. On the other hand, it is a memory whose meaning is only arrived at during the course of analysis, in the analytic experience. This is the moment at which repetition appears, which can no longer be reduced to the subject's account as an attempt to imaginatively symbolize reality and which is expressed as the pain of being in the transference, in the relationship with the analyst as other (AU)

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Slippery substrates impair ATP-dependent protease function by slowing unfolding.

ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444-1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243-13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.