SUMMARY The UCH37 deubiquitylase functions in two large and very different complexes, the 26S proteasome and the INO80 chromatin remodeler. We have performed biochemical characterization and determined crystal structures of UCH37 in complexes with RPN13 and NFRKB, which mediate its recruitment to proteasome and INO80, respectively. RPN13 and NFRKB make similar contacts to the UCH37 C-terminal domain, but quite different contacts to the catalytic UCH domain. RPN13 can activate UCH37 by disrupting dimerization, although physiologically-relevant activation likely results from stabilization of a surface competent for ubiquitin binding and modulation of the active-site crossover loop. In contrast, NFRKB inhibits UCH37 by blocking the ubiquitin-binding site and by disrupting the enzyme active site. These findings reveal remarkable commonality in mechanisms of recruitment, yet very different mechanisms of regulating enzyme activity, and provide a foundation for understanding the role of UCH37 in the unrelated proteasome and INO80 complexes.

Over 80% of diffuse intrinsic pontine gliomas (DIPGs) harbor a point mutation in histone H3.3 where lysine 27 is substituted with methionine (H3.3K27M); however, how the mutation affects kinetics and function of PcG proteins remains elusive. We demonstrate that H3.3K27M prolongs the residence time and search time of Ezh2, but has no effect on its fraction bound to chromatin. In contrast, H3.3K27M has no effect on the residence time of Cbx7, but prolongs its search time and decreases its fraction bound to chromatin. We show that increasing expression of Cbx7 inhibits the proliferation of DIPG cells and prolongs its residence time. Our results highlight that the residence time of PcG proteins directly correlates with their functions and the search time of PcG proteins is critical for regulating their genomic occupancy. Together, our data provide mechanisms in which the cancer-causing histone mutation alters the binding and search dynamics of epigenetic complexes.

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle.

In the original version of the above article, Table 2 reported that the UCH37(M148A/F149A)/RPN13 mutant complex has kinetic parameters indistinguishable from those of unbound UCH37. Further investigation of the UCH37 M148A/F149A construct revealed a gene duplication event that resulted in expression of a truncated protein product. Experiments repeated with the correct UCH37 M148A/F149A construct, in addition to a UCH37 M148D/F149D double mutant, reveal only a slight effect on the kinetic parameters. These results therefore suggest a minor role for these residues in the activation of UCH37 and in subsequent turnover of the Ub-AMC substrate. All other conclusions derived from Table 2 remain unaltered by this correction. An updated Table 2 is printed below and will be corrected in the online version of the article. The correct plasmids encoding UCH37 M148A/F149A and M148D/F149D have been submitted to the Addgene plasmid repository. The list of plasmids used in this study has been updated in the original article's Supplemental Information.

No abstract