Knowledge gains in a professional development workshop on diversity, equity, inclusion, and implicit bias in academia.

As literature indicates, historic racism and implicit bias throughout academia have been profound metrics leading to a lack of diversity, as related to people from underrepresented groups according to race and ethnicity, among biomedical sciences graduate students in U.S. universities. Recognizing such challenges, a team of biomedical scientists and inclusivity educators developed and implemented a pilot training program within an academic health sciences center as an initial step to educate faculty and staff regarding their roles in the promotion of an inclusive academic environment, receptive to all students, including underrepresented students. The 3-h workshop included didactic modules, videos, teaching modules, and active attendee participation. Faculty and staff were presented common terminology and ways to promote the development of an inclusive and diverse academic workforce. Compared with pre-workshop, post-workshop survey results indicated a statistically significant improvement in attendee knowledge of correctly identifying definitions of "implicit bias," "status leveling," "color-blind racial attitudes," "tokenism," and "failure to differentiate." Additionally, by the end of the workshop, participants had a statistically significant increase in self-perceptions regarding the importance of improving diversity and recognizing biases and stereotypes in graduate education, knowing what to say when interacting with people from different cultures, and the ability to acknowledge bias when mentoring students from groups underrepresented in the biomedical field. This preliminary initiative was successful in the introduction of faculty and staff to the importance of fostering an inclusive academic environment and thereby developing a diverse workforce.

Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly.

The NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin-binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT-ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2.

The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 µm (Ki = 2.5 ± 1.3 µm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 µm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.