CS Domains Research Articles

OR26-3 USP19 Deubiquitinating Enzyme Functions as a Novel Co-Chaperone of Hsp90 to Regulate Glucocorticoid Receptor Levels

Abstract Ubiquitin specific protease 19 (USP19) is a deubiquitinating enzyme involved in metabolism. Its expression is upregulated in skeletal muscle in many conditions of muscle wasting. Genetic inactivation of USP19 (USP19KO) in mice protects from muscle atrophy and diet induced obesity and insulin resistance. We previously reported that the protection against muscle atrophy in USP19KO is due at least in part to reduction in muscle glucocorticoid receptor (GR) levels and signaling. Here, we report that USP19KO mice show > 50% decrease in GR protein level in multiple tissues with no observed changes in GR mRNA levels. Overexpression of the endoplasmic reticulum or cytosol-localized isoforms of USP19 in cells resulted in increased GR protein levels, while knockdown by siRNA or knockout by CRISPR-Cas9 resulted in decreased GR protein levels. Intriguingly, GR was not more ubiquitinated in the absence of USP19. Furthermore, expression of a catalytically inactive form of USP19 also increased GR protein levels and exposure of cells to a small molecule inhibitor of USP19 enzymatic activity had no effects on levels of GR. Deletion analysis revealed that the N-terminal CS domains of USP19, which are structurally similar to the p23 co-chaperone of HSP90, were required for increasing levels of GR. Since HSP90 is known to regulate GR folding and stability, this suggested that USP19 may function as a p23 like co-chaperone. Indeed, overexpression of USP19 not only increased GR levels, but translocation to the nucleus and transcriptional activity of a reporter gene. Using a novel BRET assay, we observed that USP19 interacts indirectly with GR through binding with HSP90. This interaction with HSP90 is critically dependent on the CS2 domain of USP19 and can be competed by expression of the p23 co-chaperone of HSP90. We have therefore identified a novel co-chaperone-like mechanism through which USP19 regulates GR protein stability independent of its catalytic activity. Targeting the interaction of USP19 with the GR-HSP90 complex could be a strategy to decrease GR levels, reduce glucocorticoid signalling and prevent muscle atrophy. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Domain interactions reveal auto-inhibition of the deubiquitinating enzyme USP19 and its activation by HSP90 in the modulation of huntingtin aggregation.

Ubiquitin-specific protease 19 (USP19) is a member of the deubiquitinating (DUB) enzymes that catalyze removing the ubiquitin signals from target proteins. Our previous research has demonstrated that USP19 up-regulates the protein level and aggregation of polyQ-expanded huntingtin through the involvement of heat shock protein 90 (HSP90). Here, we present solution structures of the CS1, CS2 and UbL domains of USP19 and structural insights into their domain interactions. We found that the tandem CS domains fold back to interact with the C-terminal USP domain (USPD) intra-molecularly that leads to inhibition of the catalytic core of USP19, especially CS1 interacts with the embedded UbL domain and CS2 does with the CH2 catalytic core. Moreover, CS2 specifically interacts with the NBD domain of HSP90, which can activate the DUB enzyme. A mechanism of auto-inhibition of USP19 and activation by HSP90 is proposed, on which USP19 modulates the protein level of polyQ-expanded huntingtin in cells. This study provides structural and mechanistic insights into the modulation of protein level and aggregation by USP19 with the assistance of HSP90.

Cognición social en el abuso de sustancias. Una revisión narrativa

Introducción: la Cognición Social (CS) es el conjunto de procesos neurobiológicos, psicológicos y sociales que permiten percibir, reconocer y evaluar una situación social con el objetivo de generar la respuesta conductual más apropiada para cada contexto. Se integra por cinco dominios: Teoría de la Mente, Procesamiento Emocional, Estilo Atribucional, Percepción Social y Conocimiento Social. Al hablar del abuso de sustancias, la CS cobra relevancia por su implicación en el pronóstico terapéutico y la reinserción social de los usuarios en rehabilitación. Objetivo: explorar la información existente dentro de la literatura científica, en torno al funcionamiento de los dominios de CS en abusadores de sustancias. Metodo: se realizó una búsqueda de información en bases de datos digitales. Se consideraron investigaciones cuasi y experimentales, publicadas a partir del año 2000, que incluyeran la evaluación de al menos un dominio de la CS en participantes con abuso o dependencia de sustancias. Resultados: se encontraron reportes de investigación que documentan alteraciones en los dominios de Procesamiento Emocional y Teoría de la Mente, en casos de abuso de alcohol, cannabis, metanfetaminas, cocaína y opiáceos; las alteraciones tienden a variar dependiendo de la sustancia y la severidad de consumo. No se encontraron documentos que evidencien el funcionamiento de los dominios Sesgo Atribucional, Percepción Social ni Conocimiento Social. Discusión y conclusiones: resulta aventurado señalar la existencia de un déficit de CS y sus dominios para cada sustancia en específico; los estudios son aún escasos, existe variabilidad respecto a los instrumentos empleados y falta considerar otras variables clínicas que pudieran moderar o modificar la asociación entre los dominios de la CS y el consumo abusivo de sustancias.

Structural Basis for Assembly of Hsp90-Sgt1-CHORD Protein Complexes: Implications for Chaperoning of NLR Innate Immunity Receptors

SummaryHsp90-mediated function of NLR receptors in plant and animal innate immunity depends on the cochaperone Sgt1 and, at least in plants, on a cysteine- and histidine-rich domains (CHORD)-containing protein Rar1. Functionally, CHORD domains are associated with CS domains, either within the same protein, as in the mammalian melusin and Chp1, or in separate but interacting proteins, as in the plant Rar1 and Sgt1. Both CHORD and CS domains are independently capable of interacting with the molecular chaperone Hsp90 and can coexist in complexes with Hsp90. We have now determined the structure of an Hsp90-CS-CHORD ternary complex, providing a framework for understanding the dynamic nature of Hsp90-Rar1-Sgt1 complexes. Mutational and biochemical analyses define the architecture of the ternary complex that recruits nucleotide-binding leucine-rich repeat receptors (NLRs) by manipulating the structural elements to control the ATPase-dependent conformational cycle of the chaperone.

Investigation on the properties of methoxy poly(ethylene glycol)/chitosan graft co-polymers

Methoxy poly(ethylene glycol)/chitosan graft co-polymers (CS-g-mPEGs) with different degrees of substitution were synthesized by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde. The crystalline and thermal properties of CS-g-mPEGs were characterized by wide-angle X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetry (TG). The results indicate that CS-g-mPEG solids represent microphase separation morphology with mPEG crystal and CS domains coexistence and the introduction of PEG on CS improves the thermal decomposition. The hydrodynamic behavior of CS-g-mPEGs in aqueous solution and the influence of NaCl were investigated. The results indicate that the hydrodynamic behavior of CS-g-mPEGs in aqueous solution is significantly affected by the degree of substitution and the concentration of NaCl, which are quite different from that of CS. The results of this paper also certify that CS-g-mPEGs keep the property of complexation with a counter-ion, such as tripolyphosphate, to form nanoparticles through the electrostatic interaction.

The Modular Structure of SIP Facilitates Its Role in Stabilizing Multiprotein Assemblies,

Siah-interacting protein (SIP) was identified as a novel adaptor that physically links the E3 ubiquitin ligase activity of Siah-1 with Skp1 and Ebi F-Box protein in the degradation of beta-catenin, a transcriptional activator of TCF/LEF genes. In this study, we have used solution NMR spectroscopy to characterize the domain structure of SIP, which includes a novel helical hairpin domain at the N-terminus flexibly linked to a CS domain and an unstructured carboxy terminal SGS domain. These studies have been complemented by mapping the sites of functionally important protein-protein interactions involving Siah-1 and Skp1 to individual domains of SIP. NMR-based chemical shift perturbation assays show that Siah-1 interacts with the flexible linker between SIP N and CS domains. This site for interaction in the linker does not perturb residues in the structured region at the N-terminus but does appear to restrict the rotational freedom of the SIP CS domain in the context of the full-length protein. In contrast, Skp1 engages the SIP CS domain exclusively through weak interactions that are not coupled to the other domains. The principal role of the modular structure of SIP appears to be in bringing these two proteins into physical proximity and orchestrating the orientation required for polyubiquitination of beta-catenin in the intact SCF-type complex.

The Low Sulfated Chondroitin Sulfate Proteoglycans of Human Placenta Have Sulfate Group-clustered Domains That Can Efficiently Bind Plasmodium falciparum-infected Erythrocytes

Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with approximately 30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only approximately 8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2-3% and 9-14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20-28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation.

Cleavage of the Carboxyl Tail from the G3 Domain of Aggrecan but Not Versican and Identification of the Amino Acids Involved in the Degradation

Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.