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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.

OBJECTIVES/HYPOTHESIS Voice disorders in Parkinson's disease (PD) are early-onset, manifest in the preclinical stages of the disease, and negatively impact quality of life. The complete loss of function in the PTEN-induced kinase 1 gene (Pink1) causes a genetic form of early-onset, autosomal recessive PD. Modeled after the human inherited mutation, the Pink1-/- rat demonstrates significant cranial sensorimotor dysfunction including declines in ultrasonic vocalizations. However, the underlying genetics of the vocal fold thyroarytenoid (TA) muscle that may contribute to vocal deficits has not been studied. The aim of this study was to identify differentially expressed genes in the TA muscle of 8-month-old male Pink1-/- rats compared to wildtype controls. STUDY DESIGN Animal experiment with control. METHODS High throughput RNA sequencing was used to examine TA muscle gene expression in adult male Pink1-/- rats and wildtype controls. Weighted Gene Co-expression Network Analysis was used to construct co-expression modules to identify biological networks, including where Pink1 was a central node. The ENRICHR tool was used to compare this gene set to existing human gene databases. RESULTS We identified 134 annotated differentially expressed genes (P

Protein homeostasis is tightly regulated by the ubiquitin proteasome pathway. Disruption of this pathway gives rise to a host of neurological disorders. Through whole exome sequencing (WES) in families with neurodevelopmental disorders, we identified mutations in PSMD12, a core component of the proteasome, underlying a neurodevelopmental disorder with intellectual disability (ID) and features of autism spectrum disorder (ASD). We performed WES on six affected siblings from a multiplex family with ID and autistic features, the affected father, and two unaffected mothers, and a trio from a simplex family with one affected child with ID and periventricular nodular heterotopia. We identified an inherited heterozygous nonsense mutation in PSMD12 (NM_002816: c.367C>T: p.R123X) in the multiplex family and a de novo nonsense mutation in the same gene (NM_002816: c.601C>T: p.R201X) in the simplex family. PSMD12 encodes a non-ATPase regulatory subunit of the 26S proteasome. We confirm the association of PSMD12 with ID, present the first cases of inherited PSMD12 mutation, and demonstrate the heterogeneity of phenotypes associated with PSMD12 mutations.

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process that eukaryotic cells use to regulate their functions and coordinate their signaling networks. In particular, it plays a crucial role in neuronal development regulating very important functional and morphological interplays of neurons, such as synaptic plasticity, neurotransmitter release and morphogenesis of axons. Pathogenic alterations of genes involved in the proteolysis by UPS have been associated with several neurodevelopmental disorders (NDDs) and human diseases, highlighting the importance of this regulatory mechanism to developmental processes and neurogenesis. Here, we describe a 19 years old male patient showing a syndromic form of NDD. The main clinical features are intellectual disability/speech delay, congenital anomalies and facial dysmorphisms. Through a targeted resequencing approach (TRS), we identified a missense variant in PSMD12, a gene recently associated to an emerging syndromic form of NDD, which encodes for the non-ATPase subunit of the 19S regulator of 26S proteasome complex. The variant described herein, inherited from the father with apparently borderline cognitive ability, is useful to expand the molecular spectrum of heterozygous PSMD12 mutations and to provide insight into the molecular pathogenesis of this new condition since it is, to the best of our knowledge, the first missense substitution to date reported in medical literature. More importantly, our study highlight once again the utility of next generation sequencing in establishing an etiological basis in clinically and genetically heterogeneous conditions such as NDDs, thus allowing a better diagnosis, counseling and management of affected patients and their families.

Interstitial 17q24.1 or 17q24.2 deletions were reported after conventional cytogenetic analysis or chromosomal microarray analysis in patients presenting intellectual disability, facial dysmorphism, and/or malformations. We report on a fetus with craniofacial dysmorphism, talipes equinovarus, and syndactyly associated with a de novo 2.5 Mb 17q24.1q24.2 deletion. Among the deleted genes, KPNA2 and PSMD12 are discussed for the correlation with the fetal phenotype. This is the first case of prenatal diagnosis of 17q24.1q24.2 deletion.

Iako kromosom 17 pokazuje vrlo veliku genetičku nestabilnost, intersticijske delecije unutar regije 17q24 vrlo su rijetke. Bolesnici s ovom promjenom pokazuju heterogen fenotip: mikrocefaliju, tipične dismorfi čne crte, srčane grješke, intelektualno zaostajanje, Andersen-Tawilov sindrom i Carneyev kompleks. Prikazujemo 10-mjesečnu bolesnicu s mikrocefalijom, specifi čnom dismorfi jom te zaostajanjem u tjelesnom i psihomotornom razvoju, kod koje je utvrđena rijetka mikrodelecija regije 17q24.1-q24.3 veličine 5.4 Mb. Ova delecija zahvaća ukupno 30 gena koji kodiraju proteine. Smatramo da su za fenotip bolesnice vjerojatno odgovorni geni PRKCA, PSMD12, KPNA2, PRKAR1A i KCNJ2. Promjene u genu PRKCA pronađene su u osoba s poremećenom kognitivnom funk cijom, dok se haploinsufi cijencija PSMD12 povezuje s neurorazvojnim poremećajima i dismorfi jom. Delecije gena KPNA2, uklju čenog u signalni put sindroma lomljivosti Nijmegen, uzrokuju mikrocefaliju, nizak rast i sindaktiliju. Gen PRKAR1A se povezuje s nastankom Carneyevog kompleksa, a haploinsufi cijencija gena KCNJ2 uzrokuje mišićnu slabost i aritmije. Neki od ovih simptoma nisu zapaženi kod bolesnice, ali bi se, kao i niz drugih komorbiditeta, s vremenom mogli razviti, zbog čega je nužno njeno kontinuirano praćenje., Although chromosome 17 shows high genetic instability, interstitial deletions within 17q24 region are very rare. The phenotype of aff ected patients is heterogeneous but generally includes microcephaly, dysmorphic features, heart defects, intellectual disability, Andersen-Tawil syndrome and Carney complex. We present a 10-month-old proband with rare 5.4 Mb microdeletion of 17q24.1- q24.3 region. Her main clinical manifestations include microcephaly, distinctive dysmorphic features, and hypotrophy. Among 30 deleted protein coding genes, we hypothesize that PRKCA, PSMD12, KPNA2, PRKAR1A, and KCNJ2 might be responsible for the proband’s phenotype. Changes in PRKCA gene have been described in patients with abnormal cognitive function and haploinsuffi - ciency of PSMD12 has been associated with neurodevelopmental disorders and dysmorphia. Deletions of KPNA2 gene, known to be involved in Nijmegen breakage syndrome, are characterized by microcephaly, short stature, and syndactyly. PRKAR1A gene is known to be involved in Carney complex. Haploinsuffi ciency of KCNJ2 gene in this region causes episodes of muscle weakness and arrhythmia. So far, these symptoms have not been observed in the proband. Nevertheless, as these and additional comorbidities might develop over time, continuous monitoring is required.

12 páginas Trichosporon asahii es un hongo patógeno emergente reportado en la literatura médica principalmente en pacientes inmunocomprometidos. No obstante, el presente caso es inusual debido a que se trata de un paciente adulto joven inmunocompetente que presentó fungemia por T. asahii y al mismo tiempo desarrolló insuficiencia respiratoria aguda por bronquiolitis respiratoria y neumonía descamativa, la cual resolvió posterior al tratamiento antimicótico instaurado, soporte ventilatorio y vigilancia en Unidad de Cuidado Intesivo (UCI). Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

Objective To explore the candidate genes that play significant roles in the interconnection between abdominal aortic aneurysm (AAA) and type 2 diabetes mellitus (DM). Methods We used the Biomedical Discovery Support System (BITOLA) to screen out the candidate intermediate molecular (CIM) "Gene or Gene Product" that are related to AAA and DM. The dataset of GSE13760, GSE7084, GSE57691, GSE47472 were used to analyze the differentially expressed genes (DEGs) of AAA and DM compared to the healthy status. We used the online tool of Venny 2.1 assisted by manual checking to identify the overlapped DEGs with the CIMs. The Human eFP Browser was applied to examine the tissue specific expression levels of the detected genes in order to recognize strong expressed genes in both human artery and pancreatic tissue. Results There were 86 CIMs suggested by the closed BITOLA system. Among all the DEGs of AAA and DM, 8 genes in GSE7084 (ISG20, ITGAX, DSTN, CCL5, CCR5, AGTR1, CD19, CD44) and 2 genes in GSE13760 (PSMD12, FAS) were found to be overlapped with the 86 CIMs. By manual checking and comparing with tissue-specific gene data through Human eFP Browser, the gene PSMD12 (proteasome 26S subunit, non-ATPase 12) was recognized to be strongly expressed in both the aorta and pancreatic tissue. Conclusion We proposed a hypothesis through text mining that PSMD12 might be involved or potentially involved in the interconnection between AAA and DM, which may provide a new clue for studies on novel therapeutic strategies for the two diseases.

Our understanding of the transition from physiological to pathological cardiac hypertrophy remains elusive and largely based on reductionist hypotheses. Here, we profiled the translatomes of 15 mouse hearts to provide a molecular blueprint of altered gene networks in early cardiac remodeling. Using co-expression analysis, we showed how sub-networks are orchestrated into functional modules associated with pathological phenotypes. We discovered unappreciated hub genes, many undocumented for their role in cardiac hypertrophy, and genes in the transcriptional network that were rewired in the translational network, and associated with semantically different subsets of enriched functional terms, such as Fam210a, a novel musculoskeletal modulator, or Psmd12, implicated in protein quality control. Using their correlation structure, we found that transcriptome networks are only partially reproducible at the translatome level, providing further evidence of post-transcriptional control at the level of translation. Our results provide novel insights into the complexity of the organization of in vivo cardiac regulatory networks.