RESUMEN

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

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RESUMEN

TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction.

RESUMEN

Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or endoplasmic reticulum-associated degradation (ERAD). Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10, and the Asi complex, which resides at the nuclear envelope (NE). These ER/NE membrane-associated RING-type E3 ligases recognize and ubiquitylate defective proteins in cooperation with the E2 conjugating enzyme Ubc7 and the obligatory Ubc7 cofactor Cue1. Interaction of Ubc7 with the RING domains of its cognate E3 Ub-protein ligases stimulates the formation of isopeptide (amide) Ub-Ub linkages. Each isopeptide bond is formed by transfer of an Ubc7-linked activated Ub to a lysine side chain of an acceptor Ub. Multiple Ub transfer reactions form a poly-Ub chain that targets the conjugated protein for degradation by the proteasome. To study the mechanism of Ub-Ub bond formation, this reaction is reconstituted in a cell-free system consisting of recombinant E1, Ub, Ubc7, its cofactor Cue1, and the RING domain of either Doa10 or Hrd1. Here we provide detailed protocols for the purification of the required recombinant proteins and for the reactions that produce an Ub-Ub bond, specifically, the formation of an Ubc7~Ub thiolester (Ub charging) and subsequent formation of the isopeptide Ub-Ub linkage (Ub transfer). These protocols also provide a useful guideline for similar in vitro ubiquitylation reactions intended to explore the mechanism of other Ub-conjugation systems.

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RESUMEN

In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 °C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39 °C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10 h after incubation at 39 °C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay.