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1.
Mol Cell ; 69(4): 551-565.e7, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29452636

RESUMEN

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.


Asunto(s)
Aminoquinolinas/farmacología , Inflamación/prevención & control , Proteína Adaptadora de Señalización NOD2/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Sulfonas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Células Cultivadas , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores
2.
Nucleic Acids Res ; 51(20): 11277-11290, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37811893

RESUMEN

Large ribosomal subunit precursors (pre-LSUs) are primarily synthesized in the nucleolus. At an undetermined step in their assembly, they are released into the nucleoplasm. Structural models of yeast pre-LSUs at various stages of assembly have been collected using cryo-EM. However, which cryo-EM model is closest to the final nucleolar intermediate of the LSU has yet to be determined. To elucidate the mechanisms of the release of pre-LSUs from the nucleolus, we assayed effects of depleting or knocking out two yeast ribosome biogenesis factors (RiBi factors), Puf6 and Nog2, and two ribosomal proteins, uL2 and eL43. These proteins function during or stabilize onto pre-LSUs between the late nucleolar stages to early nucleoplasmic stages of ribosome biogenesis. By characterizing the phenotype of these four mutants, we determined that a particle that is intermediate between the cryo-EM model State NE1 and State NE2 likely represents the final nucleolar assembly intermediate of the LSU. We conclude that the release of the RiBi factors Nip7, Nop2 and Spb1 and the subsequent stabilization of rRNA domains IV and V may be key triggers for the release of pre-LSUs from the nucleolus.


Asunto(s)
Proteínas Ribosómicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
bioRxiv ; 2024 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-39257767

RESUMEN

Novel proteins can originate de novo from non-coding DNA and contribute to species-specific adaptations. It is challenging to conceive how de novo emerging proteins may integrate pre-existing cellular systems to bring about beneficial traits, given that their sequences are previously unseen by the cell. To address this apparent paradox, we investigated 26 de novo emerging proteins previously associated with growth benefits in yeast. Microscopy revealed that these beneficial emerging proteins preferentially localize to the endoplasmic reticulum (ER). Sequence and structure analyses uncovered a common protein organization among all ER-localizing beneficial emerging proteins, characterized by a short hydrophobic C-terminus immediately preceded by a transmembrane domain. Using genetic and biochemical approaches, we showed that ER localization of beneficial emerging proteins requires the GET and SND pathways, both of which are evolutionarily conserved and known to recognize transmembrane domains to promote post-translational ER insertion. The abundance of ER-localizing beneficial emerging proteins was regulated by conserved proteasome- and vacuole-dependent processes, through mechanisms that appear to be facilitated by the emerging proteins' C-termini. Consequently, we propose that evolutionarily conserved pathways can convergently govern the cellular processing of de novo emerging proteins with unique sequences, likely owing to common underlying protein organization patterns.

4.
Biomolecules ; 12(4)2022 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-35454122

RESUMEN

Nutrient supply dictates cell signaling changes, which in turn regulate membrane protein trafficking. To better exploit nutrients, cells relocalize membrane transporters via selective protein trafficking. Key in this reshuffling are the α-arrestins, selective protein trafficking adaptors conserved from yeast to man. α-Arrestins bind membrane proteins, controlling the ubiquitination and endocytosis of many transporters. To prevent the spurious removal of membrane proteins, α-arrestin-mediated endocytosis is kept in check through phospho-inhibition. This phospho-regulation is complex, with up to 87 phospho-sites on a single α-arrestin and many kinases/phosphatases targeting α-arrestins. To better define the signaling pathways controlling paralogous α-arrestins, Aly1 and Aly2, we screened the kinase and phosphatase deletion (KinDel) library, which is an array of all non-essential kinase and phosphatase yeast deletion strains, for modifiers of Aly-mediated phenotypes. We identified many Aly regulators, but focused our studies on the TORC1 kinase, a master regulator of nutrient signaling across eukaryotes. We found that TORC1 and its signaling effectors, the Sit4 protein phosphatase and Npr1 kinase, regulate the phosphorylation and stability of Alys. When Sit4 is lost, Alys are hyperphosphorylated and destabilized in an Npr1-dependent manner. These findings add new dimensions to our understanding of TORC1 regulation of α-arrestins and have important ramifications for cellular metabolism.


Asunto(s)
Arrestinas , Proteínas de Saccharomyces cerevisiae , Arrestina/metabolismo , Arrestinas/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteína Fosfatasa 2/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal
5.
Integr Comp Biol ; 62(2): 237-251, 2022 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-35587374

RESUMEN

The gut microbial communities of mammals provide numerous benefits to their hosts. However, given the recent development of the microbiome field, we still lack a thorough understanding of the variety of ecological and evolutionary factors that structure these communities across species. Metabarcoding is a powerful technique that allows for multiple microbial ecology questions to be investigated simultaneously. Here, we employed DNA metabarcoding techniques, predictive metagenomics, and culture-dependent techniques to inventory the gut microbial communities of several species of rodent collected from the same environment that employ different natural feeding strategies [granivorous pocket mice (Chaetodipus penicillatus); granivorous kangaroo rats (Dipodomys merriami); herbivorous woodrats (Neotoma albigula); omnivorous cactus mice (Peromyscus eremicus); and insectivorous grasshopper mice (Onychomys torridus)]. Of particular interest were shifts in gut microbial communities in rodent species with herbivorous and insectivorous diets, given the high amounts of indigestible fibers and chitinous exoskeleton in these diets, respectively. We found that herbivorous woodrats harbored the greatest microbial diversity. Granivorous pocket mice and kangaroo rats had the highest abundances of the genus Ruminococcus and highest predicted abundances of genes related to the digestion of fiber, representing potential adaptations in these species to the fiber content of seeds and the limitations to digestion given their small body size. Insectivorous grasshopper mice exhibited the greatest inter-individual variation in the membership of their microbiomes, and also exhibited the highest predicted abundances of chitin-degrading genes. Culture-based approaches identified 178 microbial isolates (primarily Bacillus and Enterococcus), with some capable of degrading cellulose and chitin. We observed several instances of strain-level diversity in these metabolic capabilities across isolates, somewhat highlighting the limitations and hidden diversity underlying DNA metabarcoding techniques. However, these methods offer power in allowing the investigation of several questions concurrently, thus enhancing our understanding of gut microbial ecology.


Asunto(s)
Microbioma Gastrointestinal , Microbiota , Animales , Quitina , Dipodomys , Herbivoria , Peromyscus , Roedores
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